HistochemicaI Journa] 24, 409-418 (1992)
Light-microscopic detection of acidic glycoconjugates with sensitized diamine procedures YOSHIFUMI
HIRABAYASHI
Department of Anatomy, Nagoya City University Medical School Nagoya 467, Japan Received 17 September 1991 and in revised form 19 November I991
Summary To enhance the efficiency and specificity of diamine methods in light microscopy, these methods were sensitized by silver enhancement in combination with trichloro(ethylene)platinate (KTP). The sensitized diamine methods consisted of a diamine (high or low iron diamine: HID or LID), KTP, borohydride reduction (BH) and a physical development (PD) sequence. The new methods have been successfully applied to routinely prepared tissue sections obtained from rat organs, such as salivary glands, stomach, colon, kidney, lung and trachea. In the tissues subjected to the sensitized diamine methods, weakly diamine-stained histological structures exhibited vivid positive reactions. The combined sensitized diamine methods and selective procedures, such as enzyme digestion and chemical modification, have substantiated that these methods were of sufficient efficiency and specificity.
Introduction To detect acidic glycoconjugates in light microscopy, several histochemical methods have been established and widely used, such as Alcian Blue pH 1.0 or 2.5 (Lev & Spicer, 1964; Scott & Dorling, 1965; Spicer et at., 1967), high- or low-iron diamine (Spicer, 1965; Lev & Spicer, 1965; Spicer et aI., 1967), aldehyde fuchsin (Spicer et al., 1967; Pearse, 1985), dialyzed iron ferricyanide (Yamada, 1987), Azure A (Spicer, 1960; Spicer et al., 1967), Acriflavine (Yamada, 1970; Pearse, 1985) and Cuprolinic Blue (Hirabayashi et al., 1987). With these methods, however, it has been difficult to visualize histological structures that contained extremely small amounts of acidic glycoconjugates, such as the basement membrane and intracellular organelles. More sensitive and efficient histochemical methods in light microscopy have been needed to visualize histological structures of lower acidic glycoconjugate contents. Recently, an attempt has been made to enhance the copper sulphide signal of Cuprolinic Blue by physical development and to detect small amounts of acidic glycoconjugates in tissues (Lorm6e et al., 1989). In this work, the retention of glycoconjugates was enhanced and the translocation of the carbohydrates was avoided during tissue preparations, since the dye was added to the fixative solution. However, the method using Cuprolinic Blue is deficient in at least two aspects: (i) the 0018-2214 9 1992 Chapman & Hall
reactivity of the dye with RNA, and (it) the lower potential of copper sulphide as a nucleus for physical development. Davies et al. (1983) developed an electronmicroscopical Gram stain by combining potassium trichloro(ethylene) platinate (KTP) and Crystal Violet for studies on bacterial cell walls. According to these authors, KTP is an anionic compound, containing platinum, that forms a water-insoluble complex with basic dyes. It is well known that platinum is a particularly heavy metal suitable as a nucleus for physical development (PD) (Jonker et al., 1969; Yamada et al., 1988). Among a number of basic dyes used for the detection of acidic gIycoconjugates, diamine (high or low iron diamine: HID or LID) is believed to be one of the most reliable stains for light microscopy (Pearse, 1985, Yamada, 1987). In this study, the present author succeeded in formulating and establishing an efficient histochemical method for acidic glycoconjugates by sequentially combining diamine (HID or LID), KTP, borohydride reduction (BH) and a PD (HID- or LID-KTP-BH-PD). From a series of histochemical experiments performed on a variety of rat tissues, the new method was found to be sufficiently sensitive and reliable for the histochemicat detection of most acidic glycoconjugates for light microscopy.
410
HIRABAYASHI safety lamp; the working solution was prepared as follows:
Materials and methods
Tissue preparation The salivary gland (submandibular and sublingual glands), stomach, colon, kidney, lung and trachea were dissected from Wistar strain adult male rats (8-10 weeks). Tissue blocks from these organs were fixed in Camoy's fluid at 4~ for 18-24 h, dehydrated in 100% ethanol, cleared in xylene and embedded in paraffin wax. Sections were cut at a thickness of 4 #m and affixed to glass slides. Dewaxed and hydrated sections were subjected to the following staining procedures.
Staining procedures of sensitized diamine (HID- or LID-KTP-BHPD) methods. Hydrated sections were: (1) Stained in the following high or low iron diamine (HID: pH 1.25-1.30; LID: pH 1.75-1.9) solutions (Spicer, 1965; Gad & Sylv6n, 1969; Sorvari & Arvilommi, 1973) at room temperature for 12-24h, or at 25-30~ for 60-120 min:
N,N '-dimethyl-m-phenylenediarnine
HID LID 120 mg 30 mg
(2HC1)
N,N'-dimethyl-p-phenylenediamine (HC1) Distilled water 40% Ferric chloride solution containing 5% Hydrochloric acid
20 mg
5 mg
50 ml 1.4 ml
50 ml 0.5 ml
(2) Washed in running tap water for 10-20 rain and in three changes of distilled water. (3) Incubated in 0.SmM potassium trichloro(ethylene) platinate (KTP: Aldrich Chemical Co., USA) in 0.05 M Clark-Lubs' boric acid-sodium hydroxide buffer (pH 8.0) (Clark & Lubs, 1916) at room temperature for 60 rain. (4) Rinsed in running tap water for 10-20 rnin and in three changes of distilled water. (5) Immersed in 0.1-0.5% sodium borohydride 1.0% disodium hydrogen phosphate (anhydrous) aqueous solution (BH) for 10-30 s. (6) Washed in running tap water for 10-20 rain and in three changes of distilled water. (7) Physically developed in a working solution (PD: a modification of Nakamura et al., 1985) at 20~ for 5-10 rain in a dark room equipped with a photographic
Solution A: 20% Aqueous gum arabic solution (45 ml) 10% Aqueous silver nitrate solution (1 ml) (The gum arabic solution was centrifuged at 12 000 r.p.m, for 60 min at 4~ and the supernatant was used.) Solution B: Distilled water (15 ml) Hydroquinone (100 mg) Citric acid (300 mg) The working solution was prepared by mixing solutions A and B in the dark room. The temperature of solutions A and B was adjusted to 20~ prior to use. (8) Rinsed in distilled water in the dark room. (9) Incubated in a diluted (1:5) photographic fixer (Super Fuji Fix: Fuji Film Co., Japan) for 2 rain in the dark room. (10) Washed in running tap water for 10-20 rain. (11) Dehydrated in a graded ethanol series. (12) Cleared in xylene and mounted in HSR (Kokusai Shiyaku Co., Japan). For controls, two procedures (HID or LID and KTP-BH-PD) were performed. To identify particular molecular species or their groups in the tissues which react to the sensitized diamine procedures, certain tissue sections were subjected to either digestion experiments with acidic glycoconjugate-degrading enzymes (chondroitinase ABC, testicular hyaluronidase and neuraminidase) or a chemical modification procedure (nitrous acid).
Enzyme digestion experiments (I) Digestion with chondroitinase ABC (a modification of Yamada et al., I982). Prior to staining with the HIDKTP-BH-PD method, sections were incubated in a chondroitinase ABC solution, I ml of which contained 100#tool Tris-HC1 buffer (pH 8.0), 6#mol sodium acetate, 20#tool bovine serum albumin, 2#tool disodium ethylenediaminetetraacetic acid, 2 #mol N-ethylmaleimide, l # m o l phenylmethanesulphonyl fluoride, 0.07 #mol pepstatin and 2.0-2.5 units of chondroitinase ABC (Seikagaku Kogyo Co., Japan) at 37~ for 3 h.
Figs 1-3. Submandibular gland stained with HID-KTP-BH-PD (Fig. 1), HID (Fig. 2) and HID-KTP-BH-PD following digestion with testicular hyaluronidase (Fig. 3). x400. In the HID-KTP-BH-PD-stained specimens (Fig. 1), the basement membrane of acini, mast cells (arrows) and connective tissues surrounding the blood vessels and secretory ducts have reacted vividly. Figs 4-6. Stomach stained with HID-KTP-BH-PD (Fig. 4), HID (Fig. 5) and HID-KTP-BH-PD following digestion with chondroitinase ABC (Fig. 6). x 300. In the HID-KTP-BH-PD-stained specimens (Fig. 4), the basement membrane in fundic glands, lamina propria, submucosal connective tissues and mast cells (arrows) have stained markedly. Figs 7-9. Kidney stained with HID-KTP-BH-PD (Fig. 7), HID (Fig. 8) and HID-KTP-BH-PD following the nitrous acid procedure (Fig. 9). x 300. In the HID-KTP-BH-PD-stained specimens (Fig. 7), the basement membranes of glomerulus, Bowman's capsules and urinary tubules are visualized distinctly. Figs 10-12. Trachea stained with HID-KTP-BH-PD (Fig. 10), HID (Fig. 11) and HID-KTP-BH-PD following digestion with testicular hyaluronidase (Fig. 12). x 300. In the HID-KTP-BH-PD-stained specimens (Fig. 10), the cartilage matrix, mast cells, surface goblet cells and tracheal mixed gland mucous acini have reacted strongly.
~i~ iil~
ii~ ii~ii i I
~ ~ i 84184
~ ~i~! i~i!~i!i~!~ii~!i~~I! ~i
~ ..... ~
Sensitized diamine methods for acidic glycoconjugates
(2) Digestion with testicular hyaluronidase (-Leppi & Stoward, I965; Pearse, 1985). Prior to staining with the HID-KTPBH-PD procedure, sections were immersed in 0.1 M phosphate buffer (pH 5.5) containing 0.5-1.0 mg ml -I testicular hyaluronidase (Type VIII; Sigma Chemical Co., USA) at 37~ for 3 h. (3) Digestion with neuraminidase (Hayano & Tanaka, 1967,. Kiyohara et al., 1974). Prior to staining with the LIDKTP-BH-PD method, sections were incubated in 0.5% potassium hydroxide in 70% ethanol at room temperature for 10 min (saponification) (Pearse, 1985) and then treated with 0.1 M phosphate buffer (pH 6.5) containing 1 unit ml -I of neuraminidase (Streptococcus sp.: Seikagaku Kogyo Co., Japan) at 37~ for 3 h. As controls for the above three enzyme digestion experiments, two procedures were used: (i) some sections were kept intact without any incubation procedures, and (ii) others were incubated in each buffer solution without enzymes under identical temperatures and for the same durations of time.
Chemical modification Nitrous acid procedure (Hirabayashi et al., 1989). Prior to staining with the HID-KTP-BH-PD procedure, certain sections were allowed to react with 0.24 M sodium nitrite in 1.8 M acetic acid at room temperature for 80-i20 min. As a control for the nitrous acid procedure, some sections were incubated in 1.8 M acetic acid solution under conditions comparable to those used for the experimental procedure.
Results The results obtained with the HID- or LID-KTP-BH-PD method In the rat organs examined, the HID-KTP-BH-PD procedure resulted in positive reactions of different intensities in a number of histological structures (Table 1, column 1); the structures deserving particular note included the basement membrane and septal connective tissues of the salivary glands (Fig. 1), the basement membrane and lamina propria connective tissues in the stomach (Fig. 4) and colon, the basement membranes subjacent to the glomerular and tubular epithelia and papillar connective tissues (Fig. 7), alveolar basement
413 membrane, bronchiolar lamina propria connective tissues and septal connective tissues of the lung, and lamina propria connective tissues of the trachea (Fig. 10). The LID-KTP-BH-PD procedure also gave rise to positive reactions of various intensities in numerous histological structures of the rat organs tested (Table 1, column 6). These structures corresponded largely to those exhibiting positive HID-KTP-BH-PD reactions (Figs 13, 16, 19). In addition, certain reactive histological structures in the kidney deserved attention. The LIDKTP-BH-PD reaction of the urinary tubular basement membrane was weaker in intensity than the corresponding HID-KTP-BH-PD reaction (Figs 7, 12; Table 1, columns 1, 6). In contrast, the former reaction of the glomerular mesangial matrix was markedly more intense than the latter reaction (Figs 7, 21). In all the histological structures tested, the HIDor LID-KTP-BH-PD procedure resulted in nearly the same intensities of the reactions, irrespective of the two different staining conditions for temperature and duration.
Results obtained with the control procedures Results with the HID or LID procedure. In all the rat organs examined, the HID or LID procedure failed to detect certain HID- or LID-KTP-BH-PD-reactive histological structures such as the basement membrane and parts of septal and lamina propria connective tissues (Table 1, columns 2, 7). To substantiate such findings, a series of micrographs are presented: the HID-stained salivary gland (Fig. 2), stomach (Fig. 5), kidney (Fig. 8) and trachea (Fig. 11), together with the LID-reactive salivary gland (Fig. 14), stomach (Fig. 17), colon (Fig. 20) and kidney (Fig. 22). In the rat organs tested, the majority of HID- or LID-KTP-BH-PD-reactive histological structures exhibited somewhat weaker HID or LID reactions (Figs 2,5, 8, 11, 14, 17,20 and 22; Table I, columns 2, 7). Results with the KTP-BH-PD procedure. In the rat organs examined, the KTP-BH-PD procedure resulted in either negative or doubtful reactions in nearly all the histological structures examined (Fig. 23).
Figs 13-15. Sublingual gland stained with LID-KTP-BH-PD (Fig. 13), LID (Fig. 14) and LID-KTP-BH-PD following digestion with neuraminidase (Fig. I5). x 300. When stained with LID-KTP-BH-PD (Fig. 13), a mast cell (arrow), mucous acini and septal connective tissues stain distinctly. Figs 16-18. Stomach stained with LID-KTP-BH-PD (Fig. I6), LID (Fig. 17) and LID-KTP-BH-PD following digestion with neuraminidase (Fig. I8). x 150. When stained with LID-KTP-BH-PD (Fig. 16), histological structures such as surface and foveotar mucins and mucous neck cells have reacted markedly. Figs 19, 20. Colon stained with LID-KTP-BH-PD (Fig. 19) and LID (Fig. 20). x 150. When stained with the LID-KTP-BH-PD procedure (Fig. 19), a series of structures such as colonic goblet cells, mast cells, lamina propria and submucosal connective tissues and intercellular spaces between smooth muscle cells have reacted vividly. Figs 21-23. Kidney stained with LID-KTP-BH-PD (Fig. 2I), LID (Fig. 22) and KTP-BH-PD (Fig. 23). x300. When stained with LID-KTP-BH-PD (Fig. 21), the glomerular basement membrane, Bowman's capsules and mesangial matrix have stained pronouncedly.
414
HIRABAYASHI
?77
7
?aTm ss
o65
?Y777
~77
~ IT
5
?~77
II
777?S,~
o >m
8
n~ II o
~-~ .o~
S~ ~ ~
-~a
a
~aa
v
aa 0
i
o
o
~~aaS
?~aa
. a
~, ?
~a
?aa
a
Saa
~
aa
?aa
S aa ~0.
S ~
b~
~
~o~
u
S~b
-~o
"~ ~ ~
~ o ~ o
~
~
~
~
-~'~ ~
~-~
....
o~.~ ~
415
Sensitized diamine methods for acidic glycoconjugates
Results obtained with the combined sensitized diamine and selective methods Results with combined sensitized diamine and enzyme digestion procedures. Digestion with chondroitinase ABC reduced in various degrees or entirely eliminated the intensity of HID-KTP-BH-PD reactions in nearly all the connective tissue structures of the rat organs, such as the septal and lamina propria connective tissues of the digestive and respiratory organs (Fig. 6), some gastrointestinal mast cells and tracheal cartilage matrix (Table 1, columns 1, 3). The effects of digestion with testicular hyaluronidase upon the HID-KTP-BH-PD reactions of the connective tissue histological structures were nearly similar in degree to those of digestion with chondroitinase ABC (Figs 3, 12; Table 1, columns 1, 4). The only difference between the effects produced by the two enzyme digestions was that testicular hyaluronidase digestion failed to affect the HID-KTP-BH-PD reactions of some gastrointestinal mast cells (Table 1, columns 3, 4). In the rat organs examined, neither digestion with chondroitinase ABC nor that with testicular hyaluronidase altered the intensity of the HID-KTP-BHPD reactions of every epithelial histological structure at all (Fig. 12, Table 1, columns 1, 3, 4). Digestion with neuraminidase notably diminished the intensity of LID-KTP-BH-PD reactions in a series of epithelial histological structures in the rat organs, such as the mucous acini of both the salivary (Fig. 15) and tracheal mixed glands, gastric surface and foveolar mucins (Fig. 18) and colonic goblet cells on the surface and in crypts (Table I, columns 6, 8). Digestion with the same enzyme caused a slight reduction in the intensity of the LID-KTP-BH-PD reactions in renal glomerular basement membrane (Table 1, columns 6, 8). In contrast, digestion with this enzyme hardly affected the intensity of the UD-KTP-BH-PD reactions in most of the connective tissue structures tested (Table 1, columns 6, 8). In the three digestion experiments with two proteoglycan- and one glycoprotein-degrading enzymes, the sensitized diamine reactions of all the epithelial and connective tissue structures in the buffer-incubated control sections were comparable in intensity to those in intact control sections. Results with combined sensitized diamine and chemical modification procedures. Treatment with nitrous acid resulted in either elimination or a marked decline in intensity of the HID-KTP-BH-PD reactions in all the basement membranes examined (Fig. 9) and the majority of mast cells, except for certain gastrointestinal cells (Table 1, columns 1, 5). The HID-KTP-BH-PD reactions of the rest of the histological structures tested were never affected by this type of chemical modification. The control treatment with acetic acid for the nitrous acid procedure failed to alter the intensity of the
HID-KTP-BH-PD reactions in the basement membranes and mast cells mentioned. Discussion
As stated in the introductory remarks of this paper, the diamine (HID and LID) methods (Spicer, 1965; Lev & Spicer, 1965; Spicer et al., 1967) are known to be precise techniques for the histochemical demonstration of acidic glycoconjugates and have been used widely in light and electron microscopy. Attempts have been made to clarify the mechanisms underlying the diamine staining reactions and the selectivity, i.e., specificity of the methods, and these have been precisely elucidated (Spicer, 1965; Lev & Spicer, 1965; Spicer et al., 1967; Gad & Sylv6n, 1969; Sorvari & Arvilommi, 1973; Reid et al., 1989). In 1983, Davies et al. developed an electronmicroscopic Gram stain by combining potassium trichloro(ethylene) platinate (KTP) with Crystal Violet. In this staining method, KTP, an anionic platinumcontaining compound, can be combined with cationic dyes such as Crystal Violet to form a water-insoluble complex (Davies et al., 1983). In view of this chemical property of KTP, it seemed possible that Crystal Violet could be replaced by another cationic dye, such as the reaction products of a diamine (HID or LID) complex with acidic glycoconjugates (Spicer, 1965; Lev & Spicer, 1965; Spicer et al., 1967). The mechanism underlying the reaction of the diamine complex and KTP has not yet been fully elucidated. Since both the diamine complex and KTP contain double bonds (C = N, C = O and C = C in the former; C = C in the latter), however, both compounds are believed to be much more chemically reactive with each other, as compared with combinations of other cationic dyes and KTP (Torii, I963; Spicer, 1965; Lev & Spicer, 1965; Moore, 1966; Jarvis et aI., 1971; Chock et al., 1973). Thus, the diamine complex is conceived to deserve a substance of choice, with which better histochemical methods using KTP can be developed for acid glycoconjugates. Incidentally, KTP (molecular formula: K[PtC13(C2H4)]/ H20) is a platinum complex which yields platinum metal if subjected to reduction with hydrogen (Torii, 1963; Moore, 1966; Jarvis et al., 1971; Chock et al., 1973). In a histochemical system, platinum can be a nucleus for physical development (silver enhancement) (Jonker et al., 1969; Yamada et al., 1988), which has been employed as an effective intensifying procedure for establishing a series of efficient histochemical methods to detect a variety of substances - such as heavy metals (Danscher, 1981; Fujimori et al., 1988b), tissue antigens (Holgate et al., 1983; Springall et al., 1984; Fujimori & Nakamura, I985), glycoconjugates (Manigley & Roth, 1985; Nakamura et al., 1985; Kitamura et al., 1988; Fujimori et al., 1988a; Yamada et al., 1988), proteins and amino acids (Yamada et al., 1990), nucleic acids (Tsukise et al., 1990) and unsaturated lipids (Fujimori et al., 1991).
416 On the basis of what has been discussed above, an efficient histochemical method has been successfully established in the present study. The new method consists of a diamine (HID or LID), KTP, BH and PD sequence. In this method, the reaction products of a diamine (HID or LID) complex with acidic glycoconjugates; can bind to KTP, and the bound KTP can be reduced by sodium borohydride to form platinum metal - which is then subjected to a physical development procedure to yield the final reaction products (metallic silver). According to the results obtained in this study, all the epithelial and connective tissue structures known to contain sulphated glycoconjugates (Spicer, 1960, 1965; Spicer et al., 1967; Kanwar & Farquhar, 1979; Tsuyama et al., 1983; van Kuppevelt et al., 1984; Enerb/ick et al., 1985; Enerb~ick, 1987; Hirabayashi et al., 1987) reacted with the HID-KTP-BH-PD method. Of the HID-KTP-BHPD-stained histological structures e:~amined, the lingual gland mucous acini of Wistar rats exhibited positive HID-KTP-BH-PD reactions. According to Reid et al. (1989), the same glandular mucous acini in SpragueDawley (SD) rats never stained with HID. Since the pH level of the HID solution used in this work was 1.25-1.30, however, it is certain that the HID-KTP-BHPD procedure could selectively stain sulphate groups only (Lev & Spicer, 1965; Pearse, 1985). When the present author tried to stain the sublingual glands of SD rats by using the HID-KTP-BH-PD procedure, the sublingual gland mucous acini likewise failed to stain - in keeping with previous results (Reid el al., 1989). Thus, the discrepancy between the two rat strains in the stainability of the same gland acini may be due to a strain difference in this rodent species. In the present study, all the epithelial and connective tissue structures containing acidic glycoconjugates (Spicer, 1960, 1965; Spicer et al., 1967; Tsuyama et al., 1983; van Kuppevelt et al., 1984; Hirabayashi r al., 1987; Holth6fer et aI., 1988; Laitinen et al., 1989) have been found to exhibit positive LID-KTP-BH-PD reactions. Incidentally, the present difference between HID- and LID-KTP-BH-PD staining intensities of renal glomerular and urinary tubular basement membranes deserves comment. According to Spicer (1965), HID stains sulphate esters only, whereas LID colours both sulphate esters and carboxyl groups. If LID stains both acidic groups, the LID-KTP-BH-PD reaction of the basement membranes must be significantly stronger in intensity than the corresponding HID-KTP-BH-PD reaction. In the urinary tubular basement membrane, however, the LIDKTP-BH-PD reaction is weaker in intensity than the HID-KTP-BH-PD reaction. Another finding of particular note was the intensity of the LID-KTP-BH-PD reaction of the sublingual mucous acini following digestion with neuraminidase. If LID stains both sulphate esters and carboxyl groups, then the LID-KTP-BH-PD reaction of the mucous acini should be either similar in intensity to
HIRABAYASHI or stronger than the corresponding HID-KTP-BH-PD reaction following digestion with neuraminidase. However, the present results did not support such a conclusion. In view of the contradictory results obtained here in terms of the selectivities of the LID and LIDKTP-BH-PD stainings, it seems likely that the LID-KTPBH-PD procedure stains primarily carboxyl groups and only parts of sulphate esters. It is of particular interest that certain HID- or LID-KTPBH-PD- reactive structures failed to stain with HID or LID and the HID- or LID-KTP-BH-PD reactions of the majority of the epithelial and connective tissue structures were of markedly stronger intensity than their HID or LID reactions. All these results appear to substantiate that the HID- or LID-KTP-BH-PD methods are of sufficient selectivity, i.e., specificity, and efficiency. In view of the established substrate specificities of chondroitinase ABC (Yamagata e~ al., 1968; Yamada et al., 1982; Hirabayashi et aI., 1987), testicular hyaluronidase (Leppi & Stoward, 1965; Spicer et al., 1967) and neuraminidase (Spicer & Warren, 1960; Hayano & Tanaka, 1967; Kiyohara et al., 1974) together with the histochemical significance of the chemical modification procedure with nitrous acid (Shively & Conrad, 1976; Hirabayashi et al., 1989), the present results can furthermore be regarded as providing concrete evidence for the sufficient selectivity, i.e., specificity, of the HID- or LID-KTP-BH-PD method in light microscopy. Taken together, the sensitized diamine methods reported here could be postulated to be a precise technique for the light-microscopic histochemical analysis of acidic glycoconjugates in general.
Acknowledgements The author expresses his sincere thanks to Professor Dr Kazuyori Yamada, Department of Anatomy, Nagoya City University Medical School, for the valuable comments and guidance during the course of the present study. This study was supported in part by a Grant in Aid for Scientific Research from the Ministry of Education, Science and Culture (No. 01480109).
References CHOCK, P. B., HALPERN, J. & PAULIK, F. E. (1973)
Potassium
trichloro(ethylene)platinate (II) (Zeise's salt). Inorg. Synth. 14, 90-2. CLARK, W. M. & LUBS, H. A. (1916) H y d r o g e n electrode potentials
of phthalate, phosphate, and borate buffer mixtures. J. Biol. Chem. 25, 479-510. DANSCHER,G. (1981) Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electron microscopy. Histochemistry 71, 1-16. DAVIES, J. A., ANDERSON, G. K., BEVERIDGE, T. J. & CLARK, H. C.
(1983) Chemical mechanism of the Gram stain and
Sensitized diamine methods for acidic glycoconjugates synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain. ]. Bacteriol. 156, 837-45. ENERBA.CKL. (1987) Mucosal mast cells in the rat and in man. Int. Archs, Allergy Appl. Immun. 82, 249-55. ENERB•CK, L., KOLSET,S. O., KUSCHE,M., HJERPE,A. & LINDAHL,U. (1985) Glycosaminoglycans in rat mucosal mast cells. Biochem. J. 227, 661-8. FUJIMORI, O. & NAKAMURA,M. (1985) Protein A gold-silver staining method for light microscopic immunohistochemistry. Arch. Histol. Jap. 48, 449-52. FUJIMORI,O., SOBUE,H. M. & YAMADA,K. (1988a) Lectin binding sites of glycoproteins as revealed by lectin-goldsilver and lectin-peroxidase-diaminobenzidine methods. Histochem. J. 20, 603-9. FUJIMORI, O., TSUKISE,A. & YAMADA,K. (1988b) Histochemical demonstration of zinc in rat epididymis using a sulphidesilver method. Histochemistry 88, 469-73. EUJIMORI, O., UEDA, H. & YAMADA, K. (1991) A histochemical method for the demonstration of unsaturated lipids. Histochem. ]. 23, 91-9. GAD, A. & SYLVgN,B. (1969) On the nature of the high iron diamine method for sulfomucins. J. Histochem. Cytochem. 17, 156-60. HAYANO, S. & TANAKA, A. (1967) Streptococcal sialidase. I. Isolation and properties of sialidase produced by group K Streptococcus. J. Bacteriol. 93, 1753-7. HIRABAYASHI,Y., KYUMA,A., KOMORI, S. & YAMADA,K. (1987) The reactions of cuprolinic blue with glycoconjugatecontaining tissues in light microscopy. Acta Histochem. Cytochem. 20, 647-58. HIRABAYASHI,Y., SHIMIZU,S. & YAMADA,K, (1989) A nitrous acid procedure as a selective histochemical means of eliminating the N-sulphates of glycoconjugates. Histochem. J. 21, 687-92. HOLGATE, C. S., JACKSON, P., COWEN, P. N. & BIRD, C. C. (1983) Immunogold~ilver staining: new method of immunostaining with enhanced sensitivity. ]. Histochem. Cytochem. 31, 938-44. HOLTHOFER, H., HENNIGAR, R. A. & SCHULTE, B. A. (1988) Glomerular sialoconjugates of developing and mature rat kidneys. Cell Differ. 24, 215-22. JARVIS, J. A. J., KILBOURAN, B. T. & OWSTON, P. G. (1971) A redetermination of the crystal and molecular structure of Zeise's salt, KPtCI3.C2H4.H20. Acta Cryst. B27, 366-72. JONKER, H., DIPPEL,C. J., HOUTMAN, H. J., JANSSEN, C. J. G. F. & VAN BEEK,L. K. H. (1969) Physical development recording systems. I. General survey and photochemical principles. Photogr. Sci. Eng. 13, 1-8. KANWAR,Y. S. & FARQUHAR,M. G. (1979) Presence of heparan sulfate in the glomerular basement membrane. Proc. NatI Acad. Sci. USA 76, 1303-7. KITAMURA,H., NAKAMUIGGM. & YAMADA,K. (1988) The utility of two physical developers for certain histochemical methods for glycoconjugates in light and electron microscopy. Proc. Kagoshima Internat. Syrup. Glycoconjugates in Medicine pp. 24-8. Tokyo: Professional Postgraduate Services. KIYOHARA, T., TERAO, T., NAKANO, K. S. & OSAWA, T. (1974) Purification and properties of a neuraminidase from Streptococcus K 6646. Arch. Biochem. Biophys. 164, 575 -82.
417 LAITINEN, L., LEHTONEN, E. & VIRTANEN, I. (1989) Differential expression of galactose and N-acetylgalactosamine residues during fetal development and postnatal maturation of rat glomeruli as revealed with lectin conjugates. Anat. Rec. 223, 311-21. LEPPI, T. J. & STOWARD, P. J. (1965) On the use of testicular hyaluronidase for identifying acid mucins in tissue sections. J. Histochem. Cytochem. 13, 406-7. LEV,R. & SPICER,S. S. (1964) Specific staining of sulphate groups with alcian blue at low pH. J. Histochem. Cytochem. 12, 309. LEV, R. & SPICER, S. S. (1965) A histochemical comparison of human epithelial mucins in normal and in hypersecretory states including pancreatic cystic fibrosis. Am. J. PathoI. 46, 23-47. LORMI~E,P., LECOLLE,S., SEPTIER,D., DENMAT,D. L. & GOLDBERG,M. (1989) Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue. J. Histochem. Cytochem. 37, 203-8. MANIGLEY,C. & ROTH,J. (1985) Applications of immunocolloids in light microscopy. IV. Use of photochemical silver staining in a simple and efficient double-staining technique. ]. Histochem. Cytochem. 33, 1247-53. MOORE, J. w. (1966) The electronic structure of K[Pt(C2H4)C13].H20. Acta Chem. Scand. 20, 1154-62. NAKAMURA,M., KITAMURA,H. & YAMADA,K. (1985) A sensitive method for the histochemical demonstration of vicinal diols of carbohydrates. Histochem. J. 17, 477-85. PEARSE, A. G. E. (1985) Carbohydrates and mucosubstances. In Histochemistry: Theoretical and Applied. Vol 2, 4th edn, pp. 675-753. Edinburgh, London, New York: Churchill Livingstone. REID, P. E., OWEN, D. A., FLETCHER,K., ROWAN, R. E., REIMER,C. L., ROUSE,G. J. & PARK C. M. (1989) The histochemical specificity of high iron diamine-alcian blue. Histochem. J. 21, 501-4. SCOTT, J. E. & DORLING, J. (1965) Differential staining of acid glycosaminoglycans (mucopolysaccharides) by alcian blue in salt solutions. Histochemie 5, 221-3. SHIVELY,J. E. & CONRAD,H. E. (1976) Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 15, 3932-42. SORVARI,T. & ARVILOMMI,H. S. (1973) Some chemical, physical and histochemical properties of three diamine fractions obtained by gel chromatography from the high-iron diamine staining solution used for localizing sulphated mucosaccharides. Histochem. J. 5, 119-30. SPICER, S. S. (1960) A correlative study of the histochemical properties of rodent acid mucopolysaccharides. J. Histochem. Cytochem. 8, 18-35. SPICER, S. S. (1965) Diamine methods for differentiating mucosubstances histochemically. ]. Histochem. Cytochem. 13, 211-34. SPICER, S. S. & WARREN,L. (1960) The histochemistry of sialic acid-containing mucoproteins. ]. Histochem. Cytochem. 8, 135-7. SPICER, S. S., HORN, R. G. & LEPPI,T. J. (1967) Histochemistry of connective tissue mucopolysaccharides. In The Connective Tissue (edited by WAGNER, B. M. & SMITH, D. E.) pp. 251-303. Baltimore: Williams & Wilkins. SPRINGALL, D. R., HACKER, G. W., GRIMELIUS, L. & POLAK, J. M. (1984) The potential of the immunogold-silver
418 staining method for paraffin sections. Histochemistry 81, 603-8. TORII, Y. (1963) Trichloroethylene platinate (II). In The Major Dictionary of Chemistry, Vol 6, (edited by MIZUSHIMA,S. et al.) pp. 461--2. Tokyo: Kyoritu-Shuppan (in Japanese). TSUKISE, A., FUJIMORI, O. & YAMADA, K. (1990) An efficient histochemical method for deoxyribonucleic acids using a silver enhancement procedure. Histochem. ]. 22, 409-15. TSUYAMA, S., SUZUKI,S. & MURATA,F. (1983) The histochemical differences of intestinal gland epithelia in the rat colon with special reference to their glycoconjugates. Acta Histochem. Cytochem. 16, 456-71. VAN KUPPEVELT, T. H. M. S. M., CREMERS, F. P. M., DOMEN, J. G. W. & KUYPER,C. M. A. (1984) Staining of proteoglycans in mouse lung alveoli. II. Characterization of the cuprolinic blue-positive, anionic sites. Histochem. J. 16, 671-86. YAMADA, K. (1970) A histochemical study on the acriflavine staining of mucopolysaccharide-containing tissues. Acta Histochem. 35, 90-101.
HIRABAYASHI YAMADA, K. (1987) Detection of carbohydrates. In (edited by YAMADA, K.) Histochemistry, Fundamentals and Practices for Beginners pp. 223-43. Tokyo: Nankodo (in Japanese). YAMADA, K., FUJITA,Y. & SIMIZU,S. (1982) The effect of digestion with keratanase (Pseudomonas sp.) on certain histochemical reactions for glycosaminoglycans in cartilaginous and corneal tissues. Histochem. ]. 14, 897-910. YAMADA, K., KITAMURA,H. & FUJIMORI,O. (1988) Applications of physical development procedures to the techniques of electron microscopic histochemistry for carbohydrates. In Histo- and CytochemMry 1988 (edited by the Japanese Society for Histo- and Cytochemistry) pp. 95-106. Tokyo: Gakusai-Kikaku (in Japanese). YAMADA, K., ABE, Y. & FUJIMORI, O. (1990) A method for histochemical demonstration of protein-bound amino groups by light microscopy. ]. Histochem. Cytochem. 38, 985 -92. YAMAGATA, T., SAITO, H., HABUCHI, O. & SUZUKI, S. (1968) Purification and properties of bacterial chondroitinases and chondrosulfatases. ]. Biol. Chem. 243, 1523-35.
Light-microscopic detection of acidic glycoconjugates with sensitized diamine procedures YOSHIFUMI
HIRABAYASHI
Department of Anatomy, Nagoya City University Medical School Nagoya 467, Japan Received 17 September 1991 and in revised form 19 November I991
Summary To enhance the efficiency and specificity of diamine methods in light microscopy, these methods were sensitized by silver enhancement in combination with trichloro(ethylene)platinate (KTP). The sensitized diamine methods consisted of a diamine (high or low iron diamine: HID or LID), KTP, borohydride reduction (BH) and a physical development (PD) sequence. The new methods have been successfully applied to routinely prepared tissue sections obtained from rat organs, such as salivary glands, stomach, colon, kidney, lung and trachea. In the tissues subjected to the sensitized diamine methods, weakly diamine-stained histological structures exhibited vivid positive reactions. The combined sensitized diamine methods and selective procedures, such as enzyme digestion and chemical modification, have substantiated that these methods were of sufficient efficiency and specificity.
Introduction To detect acidic glycoconjugates in light microscopy, several histochemical methods have been established and widely used, such as Alcian Blue pH 1.0 or 2.5 (Lev & Spicer, 1964; Scott & Dorling, 1965; Spicer et at., 1967), high- or low-iron diamine (Spicer, 1965; Lev & Spicer, 1965; Spicer et aI., 1967), aldehyde fuchsin (Spicer et al., 1967; Pearse, 1985), dialyzed iron ferricyanide (Yamada, 1987), Azure A (Spicer, 1960; Spicer et al., 1967), Acriflavine (Yamada, 1970; Pearse, 1985) and Cuprolinic Blue (Hirabayashi et al., 1987). With these methods, however, it has been difficult to visualize histological structures that contained extremely small amounts of acidic glycoconjugates, such as the basement membrane and intracellular organelles. More sensitive and efficient histochemical methods in light microscopy have been needed to visualize histological structures of lower acidic glycoconjugate contents. Recently, an attempt has been made to enhance the copper sulphide signal of Cuprolinic Blue by physical development and to detect small amounts of acidic glycoconjugates in tissues (Lorm6e et al., 1989). In this work, the retention of glycoconjugates was enhanced and the translocation of the carbohydrates was avoided during tissue preparations, since the dye was added to the fixative solution. However, the method using Cuprolinic Blue is deficient in at least two aspects: (i) the 0018-2214 9 1992 Chapman & Hall
reactivity of the dye with RNA, and (it) the lower potential of copper sulphide as a nucleus for physical development. Davies et al. (1983) developed an electronmicroscopical Gram stain by combining potassium trichloro(ethylene) platinate (KTP) and Crystal Violet for studies on bacterial cell walls. According to these authors, KTP is an anionic compound, containing platinum, that forms a water-insoluble complex with basic dyes. It is well known that platinum is a particularly heavy metal suitable as a nucleus for physical development (PD) (Jonker et al., 1969; Yamada et al., 1988). Among a number of basic dyes used for the detection of acidic gIycoconjugates, diamine (high or low iron diamine: HID or LID) is believed to be one of the most reliable stains for light microscopy (Pearse, 1985, Yamada, 1987). In this study, the present author succeeded in formulating and establishing an efficient histochemical method for acidic glycoconjugates by sequentially combining diamine (HID or LID), KTP, borohydride reduction (BH) and a PD (HID- or LID-KTP-BH-PD). From a series of histochemical experiments performed on a variety of rat tissues, the new method was found to be sufficiently sensitive and reliable for the histochemicat detection of most acidic glycoconjugates for light microscopy.
410
HIRABAYASHI safety lamp; the working solution was prepared as follows:
Materials and methods
Tissue preparation The salivary gland (submandibular and sublingual glands), stomach, colon, kidney, lung and trachea were dissected from Wistar strain adult male rats (8-10 weeks). Tissue blocks from these organs were fixed in Camoy's fluid at 4~ for 18-24 h, dehydrated in 100% ethanol, cleared in xylene and embedded in paraffin wax. Sections were cut at a thickness of 4 #m and affixed to glass slides. Dewaxed and hydrated sections were subjected to the following staining procedures.
Staining procedures of sensitized diamine (HID- or LID-KTP-BHPD) methods. Hydrated sections were: (1) Stained in the following high or low iron diamine (HID: pH 1.25-1.30; LID: pH 1.75-1.9) solutions (Spicer, 1965; Gad & Sylv6n, 1969; Sorvari & Arvilommi, 1973) at room temperature for 12-24h, or at 25-30~ for 60-120 min:
N,N '-dimethyl-m-phenylenediarnine
HID LID 120 mg 30 mg
(2HC1)
N,N'-dimethyl-p-phenylenediamine (HC1) Distilled water 40% Ferric chloride solution containing 5% Hydrochloric acid
20 mg
5 mg
50 ml 1.4 ml
50 ml 0.5 ml
(2) Washed in running tap water for 10-20 rain and in three changes of distilled water. (3) Incubated in 0.SmM potassium trichloro(ethylene) platinate (KTP: Aldrich Chemical Co., USA) in 0.05 M Clark-Lubs' boric acid-sodium hydroxide buffer (pH 8.0) (Clark & Lubs, 1916) at room temperature for 60 rain. (4) Rinsed in running tap water for 10-20 rnin and in three changes of distilled water. (5) Immersed in 0.1-0.5% sodium borohydride 1.0% disodium hydrogen phosphate (anhydrous) aqueous solution (BH) for 10-30 s. (6) Washed in running tap water for 10-20 rain and in three changes of distilled water. (7) Physically developed in a working solution (PD: a modification of Nakamura et al., 1985) at 20~ for 5-10 rain in a dark room equipped with a photographic
Solution A: 20% Aqueous gum arabic solution (45 ml) 10% Aqueous silver nitrate solution (1 ml) (The gum arabic solution was centrifuged at 12 000 r.p.m, for 60 min at 4~ and the supernatant was used.) Solution B: Distilled water (15 ml) Hydroquinone (100 mg) Citric acid (300 mg) The working solution was prepared by mixing solutions A and B in the dark room. The temperature of solutions A and B was adjusted to 20~ prior to use. (8) Rinsed in distilled water in the dark room. (9) Incubated in a diluted (1:5) photographic fixer (Super Fuji Fix: Fuji Film Co., Japan) for 2 rain in the dark room. (10) Washed in running tap water for 10-20 rain. (11) Dehydrated in a graded ethanol series. (12) Cleared in xylene and mounted in HSR (Kokusai Shiyaku Co., Japan). For controls, two procedures (HID or LID and KTP-BH-PD) were performed. To identify particular molecular species or their groups in the tissues which react to the sensitized diamine procedures, certain tissue sections were subjected to either digestion experiments with acidic glycoconjugate-degrading enzymes (chondroitinase ABC, testicular hyaluronidase and neuraminidase) or a chemical modification procedure (nitrous acid).
Enzyme digestion experiments (I) Digestion with chondroitinase ABC (a modification of Yamada et al., I982). Prior to staining with the HIDKTP-BH-PD method, sections were incubated in a chondroitinase ABC solution, I ml of which contained 100#tool Tris-HC1 buffer (pH 8.0), 6#mol sodium acetate, 20#tool bovine serum albumin, 2#tool disodium ethylenediaminetetraacetic acid, 2 #mol N-ethylmaleimide, l # m o l phenylmethanesulphonyl fluoride, 0.07 #mol pepstatin and 2.0-2.5 units of chondroitinase ABC (Seikagaku Kogyo Co., Japan) at 37~ for 3 h.
Figs 1-3. Submandibular gland stained with HID-KTP-BH-PD (Fig. 1), HID (Fig. 2) and HID-KTP-BH-PD following digestion with testicular hyaluronidase (Fig. 3). x400. In the HID-KTP-BH-PD-stained specimens (Fig. 1), the basement membrane of acini, mast cells (arrows) and connective tissues surrounding the blood vessels and secretory ducts have reacted vividly. Figs 4-6. Stomach stained with HID-KTP-BH-PD (Fig. 4), HID (Fig. 5) and HID-KTP-BH-PD following digestion with chondroitinase ABC (Fig. 6). x 300. In the HID-KTP-BH-PD-stained specimens (Fig. 4), the basement membrane in fundic glands, lamina propria, submucosal connective tissues and mast cells (arrows) have stained markedly. Figs 7-9. Kidney stained with HID-KTP-BH-PD (Fig. 7), HID (Fig. 8) and HID-KTP-BH-PD following the nitrous acid procedure (Fig. 9). x 300. In the HID-KTP-BH-PD-stained specimens (Fig. 7), the basement membranes of glomerulus, Bowman's capsules and urinary tubules are visualized distinctly. Figs 10-12. Trachea stained with HID-KTP-BH-PD (Fig. 10), HID (Fig. 11) and HID-KTP-BH-PD following digestion with testicular hyaluronidase (Fig. 12). x 300. In the HID-KTP-BH-PD-stained specimens (Fig. 10), the cartilage matrix, mast cells, surface goblet cells and tracheal mixed gland mucous acini have reacted strongly.
~i~ iil~
ii~ ii~ii i I
~ ~ i 84184
~ ~i~! i~i!~i!i~!~ii~!i~~I! ~i
~ ..... ~
Sensitized diamine methods for acidic glycoconjugates
(2) Digestion with testicular hyaluronidase (-Leppi & Stoward, I965; Pearse, 1985). Prior to staining with the HID-KTPBH-PD procedure, sections were immersed in 0.1 M phosphate buffer (pH 5.5) containing 0.5-1.0 mg ml -I testicular hyaluronidase (Type VIII; Sigma Chemical Co., USA) at 37~ for 3 h. (3) Digestion with neuraminidase (Hayano & Tanaka, 1967,. Kiyohara et al., 1974). Prior to staining with the LIDKTP-BH-PD method, sections were incubated in 0.5% potassium hydroxide in 70% ethanol at room temperature for 10 min (saponification) (Pearse, 1985) and then treated with 0.1 M phosphate buffer (pH 6.5) containing 1 unit ml -I of neuraminidase (Streptococcus sp.: Seikagaku Kogyo Co., Japan) at 37~ for 3 h. As controls for the above three enzyme digestion experiments, two procedures were used: (i) some sections were kept intact without any incubation procedures, and (ii) others were incubated in each buffer solution without enzymes under identical temperatures and for the same durations of time.
Chemical modification Nitrous acid procedure (Hirabayashi et al., 1989). Prior to staining with the HID-KTP-BH-PD procedure, certain sections were allowed to react with 0.24 M sodium nitrite in 1.8 M acetic acid at room temperature for 80-i20 min. As a control for the nitrous acid procedure, some sections were incubated in 1.8 M acetic acid solution under conditions comparable to those used for the experimental procedure.
Results The results obtained with the HID- or LID-KTP-BH-PD method In the rat organs examined, the HID-KTP-BH-PD procedure resulted in positive reactions of different intensities in a number of histological structures (Table 1, column 1); the structures deserving particular note included the basement membrane and septal connective tissues of the salivary glands (Fig. 1), the basement membrane and lamina propria connective tissues in the stomach (Fig. 4) and colon, the basement membranes subjacent to the glomerular and tubular epithelia and papillar connective tissues (Fig. 7), alveolar basement
413 membrane, bronchiolar lamina propria connective tissues and septal connective tissues of the lung, and lamina propria connective tissues of the trachea (Fig. 10). The LID-KTP-BH-PD procedure also gave rise to positive reactions of various intensities in numerous histological structures of the rat organs tested (Table 1, column 6). These structures corresponded largely to those exhibiting positive HID-KTP-BH-PD reactions (Figs 13, 16, 19). In addition, certain reactive histological structures in the kidney deserved attention. The LIDKTP-BH-PD reaction of the urinary tubular basement membrane was weaker in intensity than the corresponding HID-KTP-BH-PD reaction (Figs 7, 12; Table 1, columns 1, 6). In contrast, the former reaction of the glomerular mesangial matrix was markedly more intense than the latter reaction (Figs 7, 21). In all the histological structures tested, the HIDor LID-KTP-BH-PD procedure resulted in nearly the same intensities of the reactions, irrespective of the two different staining conditions for temperature and duration.
Results obtained with the control procedures Results with the HID or LID procedure. In all the rat organs examined, the HID or LID procedure failed to detect certain HID- or LID-KTP-BH-PD-reactive histological structures such as the basement membrane and parts of septal and lamina propria connective tissues (Table 1, columns 2, 7). To substantiate such findings, a series of micrographs are presented: the HID-stained salivary gland (Fig. 2), stomach (Fig. 5), kidney (Fig. 8) and trachea (Fig. 11), together with the LID-reactive salivary gland (Fig. 14), stomach (Fig. 17), colon (Fig. 20) and kidney (Fig. 22). In the rat organs tested, the majority of HID- or LID-KTP-BH-PD-reactive histological structures exhibited somewhat weaker HID or LID reactions (Figs 2,5, 8, 11, 14, 17,20 and 22; Table I, columns 2, 7). Results with the KTP-BH-PD procedure. In the rat organs examined, the KTP-BH-PD procedure resulted in either negative or doubtful reactions in nearly all the histological structures examined (Fig. 23).
Figs 13-15. Sublingual gland stained with LID-KTP-BH-PD (Fig. 13), LID (Fig. 14) and LID-KTP-BH-PD following digestion with neuraminidase (Fig. I5). x 300. When stained with LID-KTP-BH-PD (Fig. 13), a mast cell (arrow), mucous acini and septal connective tissues stain distinctly. Figs 16-18. Stomach stained with LID-KTP-BH-PD (Fig. I6), LID (Fig. 17) and LID-KTP-BH-PD following digestion with neuraminidase (Fig. I8). x 150. When stained with LID-KTP-BH-PD (Fig. 16), histological structures such as surface and foveotar mucins and mucous neck cells have reacted markedly. Figs 19, 20. Colon stained with LID-KTP-BH-PD (Fig. 19) and LID (Fig. 20). x 150. When stained with the LID-KTP-BH-PD procedure (Fig. 19), a series of structures such as colonic goblet cells, mast cells, lamina propria and submucosal connective tissues and intercellular spaces between smooth muscle cells have reacted vividly. Figs 21-23. Kidney stained with LID-KTP-BH-PD (Fig. 2I), LID (Fig. 22) and KTP-BH-PD (Fig. 23). x300. When stained with LID-KTP-BH-PD (Fig. 21), the glomerular basement membrane, Bowman's capsules and mesangial matrix have stained pronouncedly.
414
HIRABAYASHI
?77
7
?aTm ss
o65
?Y777
~77
~ IT
5
?~77
II
777?S,~
o >m
8
n~ II o
~-~ .o~
S~ ~ ~
-~a
a
~aa
v
aa 0
i
o
o
~~aaS
?~aa
. a
~, ?
~a
?aa
a
Saa
~
aa
?aa
S aa ~0.
S ~
b~
~
~o~
u
S~b
-~o
"~ ~ ~
~ o ~ o
~
~
~
~
-~'~ ~
~-~
....
o~.~ ~
415
Sensitized diamine methods for acidic glycoconjugates
Results obtained with the combined sensitized diamine and selective methods Results with combined sensitized diamine and enzyme digestion procedures. Digestion with chondroitinase ABC reduced in various degrees or entirely eliminated the intensity of HID-KTP-BH-PD reactions in nearly all the connective tissue structures of the rat organs, such as the septal and lamina propria connective tissues of the digestive and respiratory organs (Fig. 6), some gastrointestinal mast cells and tracheal cartilage matrix (Table 1, columns 1, 3). The effects of digestion with testicular hyaluronidase upon the HID-KTP-BH-PD reactions of the connective tissue histological structures were nearly similar in degree to those of digestion with chondroitinase ABC (Figs 3, 12; Table 1, columns 1, 4). The only difference between the effects produced by the two enzyme digestions was that testicular hyaluronidase digestion failed to affect the HID-KTP-BH-PD reactions of some gastrointestinal mast cells (Table 1, columns 3, 4). In the rat organs examined, neither digestion with chondroitinase ABC nor that with testicular hyaluronidase altered the intensity of the HID-KTP-BHPD reactions of every epithelial histological structure at all (Fig. 12, Table 1, columns 1, 3, 4). Digestion with neuraminidase notably diminished the intensity of LID-KTP-BH-PD reactions in a series of epithelial histological structures in the rat organs, such as the mucous acini of both the salivary (Fig. 15) and tracheal mixed glands, gastric surface and foveolar mucins (Fig. 18) and colonic goblet cells on the surface and in crypts (Table I, columns 6, 8). Digestion with the same enzyme caused a slight reduction in the intensity of the LID-KTP-BH-PD reactions in renal glomerular basement membrane (Table 1, columns 6, 8). In contrast, digestion with this enzyme hardly affected the intensity of the UD-KTP-BH-PD reactions in most of the connective tissue structures tested (Table 1, columns 6, 8). In the three digestion experiments with two proteoglycan- and one glycoprotein-degrading enzymes, the sensitized diamine reactions of all the epithelial and connective tissue structures in the buffer-incubated control sections were comparable in intensity to those in intact control sections. Results with combined sensitized diamine and chemical modification procedures. Treatment with nitrous acid resulted in either elimination or a marked decline in intensity of the HID-KTP-BH-PD reactions in all the basement membranes examined (Fig. 9) and the majority of mast cells, except for certain gastrointestinal cells (Table 1, columns 1, 5). The HID-KTP-BH-PD reactions of the rest of the histological structures tested were never affected by this type of chemical modification. The control treatment with acetic acid for the nitrous acid procedure failed to alter the intensity of the
HID-KTP-BH-PD reactions in the basement membranes and mast cells mentioned. Discussion
As stated in the introductory remarks of this paper, the diamine (HID and LID) methods (Spicer, 1965; Lev & Spicer, 1965; Spicer et al., 1967) are known to be precise techniques for the histochemical demonstration of acidic glycoconjugates and have been used widely in light and electron microscopy. Attempts have been made to clarify the mechanisms underlying the diamine staining reactions and the selectivity, i.e., specificity of the methods, and these have been precisely elucidated (Spicer, 1965; Lev & Spicer, 1965; Spicer et al., 1967; Gad & Sylv6n, 1969; Sorvari & Arvilommi, 1973; Reid et al., 1989). In 1983, Davies et al. developed an electronmicroscopic Gram stain by combining potassium trichloro(ethylene) platinate (KTP) with Crystal Violet. In this staining method, KTP, an anionic platinumcontaining compound, can be combined with cationic dyes such as Crystal Violet to form a water-insoluble complex (Davies et al., 1983). In view of this chemical property of KTP, it seemed possible that Crystal Violet could be replaced by another cationic dye, such as the reaction products of a diamine (HID or LID) complex with acidic glycoconjugates (Spicer, 1965; Lev & Spicer, 1965; Spicer et al., 1967). The mechanism underlying the reaction of the diamine complex and KTP has not yet been fully elucidated. Since both the diamine complex and KTP contain double bonds (C = N, C = O and C = C in the former; C = C in the latter), however, both compounds are believed to be much more chemically reactive with each other, as compared with combinations of other cationic dyes and KTP (Torii, I963; Spicer, 1965; Lev & Spicer, 1965; Moore, 1966; Jarvis et aI., 1971; Chock et al., 1973). Thus, the diamine complex is conceived to deserve a substance of choice, with which better histochemical methods using KTP can be developed for acid glycoconjugates. Incidentally, KTP (molecular formula: K[PtC13(C2H4)]/ H20) is a platinum complex which yields platinum metal if subjected to reduction with hydrogen (Torii, 1963; Moore, 1966; Jarvis et al., 1971; Chock et al., 1973). In a histochemical system, platinum can be a nucleus for physical development (silver enhancement) (Jonker et al., 1969; Yamada et al., 1988), which has been employed as an effective intensifying procedure for establishing a series of efficient histochemical methods to detect a variety of substances - such as heavy metals (Danscher, 1981; Fujimori et al., 1988b), tissue antigens (Holgate et al., 1983; Springall et al., 1984; Fujimori & Nakamura, I985), glycoconjugates (Manigley & Roth, 1985; Nakamura et al., 1985; Kitamura et al., 1988; Fujimori et al., 1988a; Yamada et al., 1988), proteins and amino acids (Yamada et al., 1990), nucleic acids (Tsukise et al., 1990) and unsaturated lipids (Fujimori et al., 1991).
416 On the basis of what has been discussed above, an efficient histochemical method has been successfully established in the present study. The new method consists of a diamine (HID or LID), KTP, BH and PD sequence. In this method, the reaction products of a diamine (HID or LID) complex with acidic glycoconjugates; can bind to KTP, and the bound KTP can be reduced by sodium borohydride to form platinum metal - which is then subjected to a physical development procedure to yield the final reaction products (metallic silver). According to the results obtained in this study, all the epithelial and connective tissue structures known to contain sulphated glycoconjugates (Spicer, 1960, 1965; Spicer et al., 1967; Kanwar & Farquhar, 1979; Tsuyama et al., 1983; van Kuppevelt et al., 1984; Enerb/ick et al., 1985; Enerb~ick, 1987; Hirabayashi et al., 1987) reacted with the HID-KTP-BH-PD method. Of the HID-KTP-BHPD-stained histological structures e:~amined, the lingual gland mucous acini of Wistar rats exhibited positive HID-KTP-BH-PD reactions. According to Reid et al. (1989), the same glandular mucous acini in SpragueDawley (SD) rats never stained with HID. Since the pH level of the HID solution used in this work was 1.25-1.30, however, it is certain that the HID-KTP-BHPD procedure could selectively stain sulphate groups only (Lev & Spicer, 1965; Pearse, 1985). When the present author tried to stain the sublingual glands of SD rats by using the HID-KTP-BH-PD procedure, the sublingual gland mucous acini likewise failed to stain - in keeping with previous results (Reid el al., 1989). Thus, the discrepancy between the two rat strains in the stainability of the same gland acini may be due to a strain difference in this rodent species. In the present study, all the epithelial and connective tissue structures containing acidic glycoconjugates (Spicer, 1960, 1965; Spicer et al., 1967; Tsuyama et al., 1983; van Kuppevelt et al., 1984; Hirabayashi r al., 1987; Holth6fer et aI., 1988; Laitinen et al., 1989) have been found to exhibit positive LID-KTP-BH-PD reactions. Incidentally, the present difference between HID- and LID-KTP-BH-PD staining intensities of renal glomerular and urinary tubular basement membranes deserves comment. According to Spicer (1965), HID stains sulphate esters only, whereas LID colours both sulphate esters and carboxyl groups. If LID stains both acidic groups, the LID-KTP-BH-PD reaction of the basement membranes must be significantly stronger in intensity than the corresponding HID-KTP-BH-PD reaction. In the urinary tubular basement membrane, however, the LIDKTP-BH-PD reaction is weaker in intensity than the HID-KTP-BH-PD reaction. Another finding of particular note was the intensity of the LID-KTP-BH-PD reaction of the sublingual mucous acini following digestion with neuraminidase. If LID stains both sulphate esters and carboxyl groups, then the LID-KTP-BH-PD reaction of the mucous acini should be either similar in intensity to
HIRABAYASHI or stronger than the corresponding HID-KTP-BH-PD reaction following digestion with neuraminidase. However, the present results did not support such a conclusion. In view of the contradictory results obtained here in terms of the selectivities of the LID and LIDKTP-BH-PD stainings, it seems likely that the LID-KTPBH-PD procedure stains primarily carboxyl groups and only parts of sulphate esters. It is of particular interest that certain HID- or LID-KTPBH-PD- reactive structures failed to stain with HID or LID and the HID- or LID-KTP-BH-PD reactions of the majority of the epithelial and connective tissue structures were of markedly stronger intensity than their HID or LID reactions. All these results appear to substantiate that the HID- or LID-KTP-BH-PD methods are of sufficient selectivity, i.e., specificity, and efficiency. In view of the established substrate specificities of chondroitinase ABC (Yamagata e~ al., 1968; Yamada et al., 1982; Hirabayashi et aI., 1987), testicular hyaluronidase (Leppi & Stoward, 1965; Spicer et al., 1967) and neuraminidase (Spicer & Warren, 1960; Hayano & Tanaka, 1967; Kiyohara et al., 1974) together with the histochemical significance of the chemical modification procedure with nitrous acid (Shively & Conrad, 1976; Hirabayashi et al., 1989), the present results can furthermore be regarded as providing concrete evidence for the sufficient selectivity, i.e., specificity, of the HID- or LID-KTP-BH-PD method in light microscopy. Taken together, the sensitized diamine methods reported here could be postulated to be a precise technique for the light-microscopic histochemical analysis of acidic glycoconjugates in general.
Acknowledgements The author expresses his sincere thanks to Professor Dr Kazuyori Yamada, Department of Anatomy, Nagoya City University Medical School, for the valuable comments and guidance during the course of the present study. This study was supported in part by a Grant in Aid for Scientific Research from the Ministry of Education, Science and Culture (No. 01480109).
References CHOCK, P. B., HALPERN, J. & PAULIK, F. E. (1973)
Potassium
trichloro(ethylene)platinate (II) (Zeise's salt). Inorg. Synth. 14, 90-2. CLARK, W. M. & LUBS, H. A. (1916) H y d r o g e n electrode potentials
of phthalate, phosphate, and borate buffer mixtures. J. Biol. Chem. 25, 479-510. DANSCHER,G. (1981) Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electron microscopy. Histochemistry 71, 1-16. DAVIES, J. A., ANDERSON, G. K., BEVERIDGE, T. J. & CLARK, H. C.
(1983) Chemical mechanism of the Gram stain and
Sensitized diamine methods for acidic glycoconjugates synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain. ]. Bacteriol. 156, 837-45. ENERBA.CKL. (1987) Mucosal mast cells in the rat and in man. Int. Archs, Allergy Appl. Immun. 82, 249-55. ENERB•CK, L., KOLSET,S. O., KUSCHE,M., HJERPE,A. & LINDAHL,U. (1985) Glycosaminoglycans in rat mucosal mast cells. Biochem. J. 227, 661-8. FUJIMORI, O. & NAKAMURA,M. (1985) Protein A gold-silver staining method for light microscopic immunohistochemistry. Arch. Histol. Jap. 48, 449-52. FUJIMORI,O., SOBUE,H. M. & YAMADA,K. (1988a) Lectin binding sites of glycoproteins as revealed by lectin-goldsilver and lectin-peroxidase-diaminobenzidine methods. Histochem. J. 20, 603-9. FUJIMORI, O., TSUKISE,A. & YAMADA,K. (1988b) Histochemical demonstration of zinc in rat epididymis using a sulphidesilver method. Histochemistry 88, 469-73. EUJIMORI, O., UEDA, H. & YAMADA, K. (1991) A histochemical method for the demonstration of unsaturated lipids. Histochem. ]. 23, 91-9. GAD, A. & SYLVgN,B. (1969) On the nature of the high iron diamine method for sulfomucins. J. Histochem. Cytochem. 17, 156-60. HAYANO, S. & TANAKA, A. (1967) Streptococcal sialidase. I. Isolation and properties of sialidase produced by group K Streptococcus. J. Bacteriol. 93, 1753-7. HIRABAYASHI,Y., KYUMA,A., KOMORI, S. & YAMADA,K. (1987) The reactions of cuprolinic blue with glycoconjugatecontaining tissues in light microscopy. Acta Histochem. Cytochem. 20, 647-58. HIRABAYASHI,Y., SHIMIZU,S. & YAMADA,K, (1989) A nitrous acid procedure as a selective histochemical means of eliminating the N-sulphates of glycoconjugates. Histochem. J. 21, 687-92. HOLGATE, C. S., JACKSON, P., COWEN, P. N. & BIRD, C. C. (1983) Immunogold~ilver staining: new method of immunostaining with enhanced sensitivity. ]. Histochem. Cytochem. 31, 938-44. HOLTHOFER, H., HENNIGAR, R. A. & SCHULTE, B. A. (1988) Glomerular sialoconjugates of developing and mature rat kidneys. Cell Differ. 24, 215-22. JARVIS, J. A. J., KILBOURAN, B. T. & OWSTON, P. G. (1971) A redetermination of the crystal and molecular structure of Zeise's salt, KPtCI3.C2H4.H20. Acta Cryst. B27, 366-72. JONKER, H., DIPPEL,C. J., HOUTMAN, H. J., JANSSEN, C. J. G. F. & VAN BEEK,L. K. H. (1969) Physical development recording systems. I. General survey and photochemical principles. Photogr. Sci. Eng. 13, 1-8. KANWAR,Y. S. & FARQUHAR,M. G. (1979) Presence of heparan sulfate in the glomerular basement membrane. Proc. NatI Acad. Sci. USA 76, 1303-7. KITAMURA,H., NAKAMUIGGM. & YAMADA,K. (1988) The utility of two physical developers for certain histochemical methods for glycoconjugates in light and electron microscopy. Proc. Kagoshima Internat. Syrup. Glycoconjugates in Medicine pp. 24-8. Tokyo: Professional Postgraduate Services. KIYOHARA, T., TERAO, T., NAKANO, K. S. & OSAWA, T. (1974) Purification and properties of a neuraminidase from Streptococcus K 6646. Arch. Biochem. Biophys. 164, 575 -82.
417 LAITINEN, L., LEHTONEN, E. & VIRTANEN, I. (1989) Differential expression of galactose and N-acetylgalactosamine residues during fetal development and postnatal maturation of rat glomeruli as revealed with lectin conjugates. Anat. Rec. 223, 311-21. LEPPI, T. J. & STOWARD, P. J. (1965) On the use of testicular hyaluronidase for identifying acid mucins in tissue sections. J. Histochem. Cytochem. 13, 406-7. LEV,R. & SPICER,S. S. (1964) Specific staining of sulphate groups with alcian blue at low pH. J. Histochem. Cytochem. 12, 309. LEV, R. & SPICER, S. S. (1965) A histochemical comparison of human epithelial mucins in normal and in hypersecretory states including pancreatic cystic fibrosis. Am. J. PathoI. 46, 23-47. LORMI~E,P., LECOLLE,S., SEPTIER,D., DENMAT,D. L. & GOLDBERG,M. (1989) Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue. J. Histochem. Cytochem. 37, 203-8. MANIGLEY,C. & ROTH,J. (1985) Applications of immunocolloids in light microscopy. IV. Use of photochemical silver staining in a simple and efficient double-staining technique. ]. Histochem. Cytochem. 33, 1247-53. MOORE, J. w. (1966) The electronic structure of K[Pt(C2H4)C13].H20. Acta Chem. Scand. 20, 1154-62. NAKAMURA,M., KITAMURA,H. & YAMADA,K. (1985) A sensitive method for the histochemical demonstration of vicinal diols of carbohydrates. Histochem. J. 17, 477-85. PEARSE, A. G. E. (1985) Carbohydrates and mucosubstances. In Histochemistry: Theoretical and Applied. Vol 2, 4th edn, pp. 675-753. Edinburgh, London, New York: Churchill Livingstone. REID, P. E., OWEN, D. A., FLETCHER,K., ROWAN, R. E., REIMER,C. L., ROUSE,G. J. & PARK C. M. (1989) The histochemical specificity of high iron diamine-alcian blue. Histochem. J. 21, 501-4. SCOTT, J. E. & DORLING, J. (1965) Differential staining of acid glycosaminoglycans (mucopolysaccharides) by alcian blue in salt solutions. Histochemie 5, 221-3. SHIVELY,J. E. & CONRAD,H. E. (1976) Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 15, 3932-42. SORVARI,T. & ARVILOMMI,H. S. (1973) Some chemical, physical and histochemical properties of three diamine fractions obtained by gel chromatography from the high-iron diamine staining solution used for localizing sulphated mucosaccharides. Histochem. J. 5, 119-30. SPICER, S. S. (1960) A correlative study of the histochemical properties of rodent acid mucopolysaccharides. J. Histochem. Cytochem. 8, 18-35. SPICER, S. S. (1965) Diamine methods for differentiating mucosubstances histochemically. ]. Histochem. Cytochem. 13, 211-34. SPICER, S. S. & WARREN,L. (1960) The histochemistry of sialic acid-containing mucoproteins. ]. Histochem. Cytochem. 8, 135-7. SPICER, S. S., HORN, R. G. & LEPPI,T. J. (1967) Histochemistry of connective tissue mucopolysaccharides. In The Connective Tissue (edited by WAGNER, B. M. & SMITH, D. E.) pp. 251-303. Baltimore: Williams & Wilkins. SPRINGALL, D. R., HACKER, G. W., GRIMELIUS, L. & POLAK, J. M. (1984) The potential of the immunogold-silver
418 staining method for paraffin sections. Histochemistry 81, 603-8. TORII, Y. (1963) Trichloroethylene platinate (II). In The Major Dictionary of Chemistry, Vol 6, (edited by MIZUSHIMA,S. et al.) pp. 461--2. Tokyo: Kyoritu-Shuppan (in Japanese). TSUKISE, A., FUJIMORI, O. & YAMADA, K. (1990) An efficient histochemical method for deoxyribonucleic acids using a silver enhancement procedure. Histochem. ]. 22, 409-15. TSUYAMA, S., SUZUKI,S. & MURATA,F. (1983) The histochemical differences of intestinal gland epithelia in the rat colon with special reference to their glycoconjugates. Acta Histochem. Cytochem. 16, 456-71. VAN KUPPEVELT, T. H. M. S. M., CREMERS, F. P. M., DOMEN, J. G. W. & KUYPER,C. M. A. (1984) Staining of proteoglycans in mouse lung alveoli. II. Characterization of the cuprolinic blue-positive, anionic sites. Histochem. J. 16, 671-86. YAMADA, K. (1970) A histochemical study on the acriflavine staining of mucopolysaccharide-containing tissues. Acta Histochem. 35, 90-101.
HIRABAYASHI YAMADA, K. (1987) Detection of carbohydrates. In (edited by YAMADA, K.) Histochemistry, Fundamentals and Practices for Beginners pp. 223-43. Tokyo: Nankodo (in Japanese). YAMADA, K., FUJITA,Y. & SIMIZU,S. (1982) The effect of digestion with keratanase (Pseudomonas sp.) on certain histochemical reactions for glycosaminoglycans in cartilaginous and corneal tissues. Histochem. ]. 14, 897-910. YAMADA, K., KITAMURA,H. & FUJIMORI,O. (1988) Applications of physical development procedures to the techniques of electron microscopic histochemistry for carbohydrates. In Histo- and CytochemMry 1988 (edited by the Japanese Society for Histo- and Cytochemistry) pp. 95-106. Tokyo: Gakusai-Kikaku (in Japanese). YAMADA, K., ABE, Y. & FUJIMORI, O. (1990) A method for histochemical demonstration of protein-bound amino groups by light microscopy. ]. Histochem. Cytochem. 38, 985 -92. YAMAGATA, T., SAITO, H., HABUCHI, O. & SUZUKI, S. (1968) Purification and properties of bacterial chondroitinases and chondrosulfatases. ]. Biol. Chem. 243, 1523-35.
