Acta anat. 94: 449-456 (1976)
Histochemistry of the duodenal glands of the cat and horse D. O d u o r -O kelo Institut für Tieranatomie der Universität Bern, Bern, Switzerland
Key words. Histochemistry ■Duodenal glands • Cat • Horse Abstract. The duodenal glands of cat and horse were studied using PAS, Alcian blue, dialysed iron, aldehyde fuchsin-Alcian blue and high iron diamine stains. It was found that the duodenal glands of the horse reacted positively to Alcian blue, dialysed iron stains and also took the Alcian blue stain in the combined aldehyde fuchsin-Alcian blue and high iron diamine-Alcian blue stains. Those of the cat gave negative results. These results suggest the presence of acidic groups in the mucosubstances secreted by the horse's duodenal glands. A suggestion is put forward on the strength of the high iron diamine-Alcian blue combined stains that the acidity is due to the presence of carboxyl groups. It is suggested that the acidity may be significant in either the cellulose metabolism or the digestion of the bacterial microflora from the stomach of herbivores.
Introduction
Received: March 29, 1975.
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Extensive studies have been carried out on the duodenal (Brunner’s) glands of various species of mammals. Bensley [1903] established the view that pyloric glands and the duo denal glands are mucous in nature. He found that mucicarmine and mucihaemalin stained the secretory masses within the cells in all the 19 species lie studied. However, F lorf.y and H arding [1934] reported distinct species variations. They obtained good mucous reactions in the goat, pig, rabbit and guinea pig. but none at all in the cat, dog and rat. Belanger [1963], applying Hale's [H ale, 1946] and Alcian blue staining techniques, reported positive reactions in the duodenal glands of the guinea pig, domestic pig, sheep and ox, but nega tive reactions in those of the rat and man. This study was carried out to compare the reactions to Alcian blue and Hale’s dialysed iron methods between the duodenal glands of the cat and horse and then to ascertain whether the positive reactions obtained were due to carboxyl or sulphate groups present in the mucous secretion.
450
O duor -O kelo
Fig. I. Photomicrograph of the duodenal portion of the cat intestine. The goblet cells show a positive reaction. Epon-embedded block. Alcian blue, pH 2.5. x 140. Fig. 2. Photomicrograph of the duodenal portion of the horse intestine. Both the goblet cells and Brunner’s glands reacted positively giving a bluish-green colour. Paraffin-em bedded block. Alcian blue, pH. 2.5. * 250.
The duodenal samples were obtained from six cats and four horses. Small pieces, fixed in Bouin's fluid or 10% formalin, were embedded in paraffin and cut on a microtome as usual. After dewaxing and bringing the sections to water, they were stained in periodic acidSchifT (PAS) reagent after M c M anus [1946], Alcian blue after Steedman [1950], Alcian blue in different concentration of magnesium chloride after S cott and D ori.ino [1965], Alcian blue-PAS after Mowry [1963], mixed diamine solutions after S picer [1965] and aldehyde fuchsin-Alcian blue sequence after S picer and M eyer [I960], Small pieces from the cat duodenum were fixed in 2.5% giuteraldehyde, osmicated and finally embedded in Epon. Thick sections were stained in PAS solutions after N evalainen et al. [1972] without
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
Materia! and methods
Histochemistry of the duodenal glands of the cat and horse
451
Fig.3. Photomicrograph of the cat duodenum. The goblet cells gave a positive blue colour but Brunner’s glands gave the magenta colour of PAS. Epon-embedded block. Combined Alcian bluc-PAS stain, x 140. Fig.4. Photomicrograph of the horse duodenum. Both the goblet cells and Brunner's glands gave the positive bluish-purple colour. Paraffin-embedded block. Combined Alcian blue-PAS stain, x 140.
prior removal of Epon. After removal of Epon, using 20% NaOH in absolute alcohol, some slides were stained in Alcian blue solutions.
In both species the goblet cells and the duodenal glands gave PAS-positive results. While the goblet cells in both species gave positive Alcian blue and Hale’s [H ale , 1946] stains, only the duodenal glands of the horse gave positive stains to both Alcian blue and Hale's techniques (fig. 1,2). In the combined Alcian blue-PAS or dialysed iron-PAS stains the duodenal glands of the cat gave positive reactions only to PAS, while those of the horse stained a positive pale to dark-blue colour (fig. 3,4).
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
Results
452
O duor /O kelo
When the sections were stained in aldehyde fuchsin-Alcian blue sequence, the goblet cells in both cat and horse exhibited a dark-blue colour. The duodenal glands of the horse took the blue colour of Alcian blue while those of the cat did not take any colour (fig. 5,6). The mixed diamine-NaCI stain revealed positive black stains only in the goblet cells in both species. With the high iron diamine-Alcian blue sequence the golbet cells ex hibited the positive black stain, but only the duodenal glands of the horse took the Alcian blue stain: those in the cat were unstained (fig.7.8).
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Fig.5. Photomicrograph of the cat duodenum. The goblet cells are clearly positive, while Brunner's glands are not. Paraffin section. Aldehyde fuchsin. x 140. Fig.6. Photomicrograph of the horse duodenum. The goblet cells gave a bluish-purple colour, while Brunner's glands gave a greenish colour. Paraffin section. Combined aldehyde fuchsin-Alcian blue stain, x 140.
Histochemistry of the duodenal glands of the cat and horse
453
Fig. 7. Photomicrograph of the cat duodenum. The goblet cells gave a positive brownishblack colour, while Brunner's glands remained uncolourcd. Paraffin sections. Combined high iron diamine-Alcian blue stain, x 140. Fig.8. Photomicrograph of the horse duodenum. The goblet cells gave a positive brownish-black colour, while Brunner's glands took a greenish-blue colour. Paraffin section. Combined high iron diamine-Alcian blue stain, x 140.
The two types of animals used in this study belong to two different mam malian orders: the cat belonging to the order Carnivora and the horse to the order Perissodactyla. The two mammals have entirely different feeding habits. It is revealed in this histochemical study that the duodenal glands in the two mammalian species react differently to Alcian blue, dialysed iron, alde hyde fuchsin and the diamine stains. Alcian blue and dialysed iron stains are commonly used in histochemistry to identify acidic mucosubstances [P earse , 1968], while combinations of aldehyde fuchsin-Alcian blue stains
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
Discussion
454
O duor -O kelo
1965], Alcian blue, pH 1.0, and Alcian blue in various electrolyte concen trations [Scott and D orling , 1965] are generally employed in histochemistry to distinguish between sulphate (-SO.,)- and carboxyl (-COOH)- containing mucosubstances [Pearse, 1968, app. 10], The results obtained in this study show that the secretions of the duodenal glands in the horse are acidic, while those of the cat are neutral. It is further shown that the nature of acidity of the mucosubstances from the duodenal glands is different from that of the goblet cell mucosubstances. The goblet cells in both species reacted positively to aldehyde fuchsin and high iron diamine, indicating the presence of sulphate groups. When these stains were followed by Alcian blue, pH 2.5, only the duodenal glands of the horse took the blue stain, indicating the presence of carboxyl groups. Similar findings have been reported by Belanger [1963] who found that the duodenal glands of the guinea pig, domestic pig, sheep and ox, were positive to Alcian blue and dialysed iron stains while those of man and rat were negative. However, Belanger [1963] did not differentiate between sulphated and carboxylated acidic mucosubstances. Autoradiographic studies by C urran and Kennedy [1955], K ent et al. [1956], J ennings and F lorey [1956] lend support to the findings in this study. These authors reported that, whereas the goblet cells in the intestinal tract of all the animals they studied were able to incorporate sulphur, there were distinct species variations with regards to Brunner's glands. J ennings and F lorey [1956] reported that in the mouse, rat and cat there was no autoradiographic incorporation of sulphur by Brunner's glands, while there was a strong to weak radioactivity in those of the guinea pig and rabbit, respectively. C urean and K ennedy [1955] stated that the auto radiographic presence of :i5S in mouse tissues was invariably associated with mucopolysaccharides and the highest activities were in those cells which produce mucopolysaccharides, e.g. intestinal goblet cells, cartilage cells and mast cells. Spicer and M eyer [I960] have reported that both superficial and deep Brunner's glands in the guinea pig reacted positively to aldehyde fuchsin, indicating the presence of sulphated mucopolysaccharide secretions. In this study the duodenal glands of the horse did not give positive reactions with either aldehyde fuchsin or high iron diamine stains. However, it is concluded that the acidity of the mucopolysaccharide secretions in the duodenal gland of the horse may be due to the presence of carboxyl groups. The significance of the species variations in the nature of secretions of the duodenal glands is not yet clear. The acidity of the mucosubstances,
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
[Spicer and M eyer, 1950], high iron diamine-AIcian blue stain [Spicer,
Histochemistry of the duodenal glands of the cat and horse
455
which may be due to carboxyl groups in the horse, but may be due to both carboxyl and sulphate groups in the guinea pig, might well be correlated to the differences in the feeding habits of the various species. The findings in this study and in previous ones seem to show that those animals which feed on cellulose-containing substances (herbivores) secrete acidic mucosubstances from their duodenal glands. These groups of animals also tend to have much more extensive development of the duodenal glands than those animals in which the duodenal secretions are neutral. It is finally postulated that the acidic mucosubstances secreted by the duodenal glands of herbivores may play an important role either in end-products of cellulose metabolism or in the metabolism of microfloral (bacterial) proteins especially in the case of ruminants. This postulation will require more histochemical and biochemical analysis. A cknowledgements This work was made possible through the Swiss Federal Government scholarship. I wish to express my sincere gratitude to Prof. Dr .W. Mosimann who offered me the facil ities in his Institute. My thanks go to Dr. R. Leiser for useful discussions and constant encouragement. I am greatly indected to Mrs. H .K opf, Miss B.H ermansson, Miss P. ROedi, Mr. U .I ff and Mr. E. G asser for their invaluable technical assistance. I should also like to thank Miss A. Ikua of the University of Nairobi who agreed to type the final script.
References Belanger, L. F .: Comparison between different histochemical and histophysical techniques
as applied to mucus-secreting cells. Ann. N.Y. Acad. Sci. 106: 364-378 (1963). Bensley. R.R.: Decennial Pubis Univ. Chicago 10: 279(1903). C urran . R.C. and K ennedy, J.S.: The distribution of the sulfated mucopolysaccharides
in the mouse. J. Path. Bact. 70: 449-457 (1955). F lorey, H.W . and H arding , H .E .: Further observations on the secretion of Brunner's
glands. J. Path. Bact. 39: 255-276 (1934). H ale, C. W.: Histochemical demonstration of acid polysaccharides in animal tissues.
Nature, Lond. 157: 802(1946). J ennings, M.A. and F lorey, H.W .: Autoradiographic observations on the mucous cells
of the stomach and intestines. Q. Jl exp. Physiol. 41: 131-151 (1956). M c M anus, J.F.A .: Histological demonstration of mucin after periodic acid. Nature,
Lond. 158: 202 (1946). M owry , R. W.: The special value of methods that colour both acidic and vicinal hydroxyl
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
groups in the histochemical iron stain, the use of Alcian blue 8 G x and their combi nations with the periodic acid-Schiff reaction, Ann. N.Y. Acad. Sci. 106:402-423 (1963).
456
O duor /O kelo
N evalainen, T.J.; Laitio, M., and Lindcren , I.: Periodic acid-SehilT (PAS) staining of
Epon-embedded tissue for light microscopy. Acta histocheni. 42: 230-233 (1972). P earse, A .G .E.: Histochemistry: theoretical and applied; 3rd ed., vol. 1 (Churchill.
London 1968). Scott . J.E . and D orlinci, J.: Differential staining of acid glycosaminoglycans (muco
polysaccharides) by Alcian blue in salt solutions. Histochemie 5: 221-233 (1965). S picer, S.S.: Diamine methods for differentiating mucosubstances histochemically. J.
Histocheni. Cytochcm. 13: 211 234 (1965). S picer, S.S. and M eyer. D. B.: Histochcmical differentiation of acid mucopolysaccharides
D. O duor-O kelo, The University of Nairobi, Department of Veterinary Anatomy, PO Box 30197, Nairobi (Kenya)
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
by means of combined aldehyde fuchsin-Alcian blue staining. Am. J. clin. Path. 33: 453-460(1960). S teedman, H .F.: Alcian blue 8 GS: a new stain for mucins. Q. Jl microsc. Sci. 91: 447449(1950).
Histochemistry of the duodenal glands of the cat and horse D. O d u o r -O kelo Institut für Tieranatomie der Universität Bern, Bern, Switzerland
Key words. Histochemistry ■Duodenal glands • Cat • Horse Abstract. The duodenal glands of cat and horse were studied using PAS, Alcian blue, dialysed iron, aldehyde fuchsin-Alcian blue and high iron diamine stains. It was found that the duodenal glands of the horse reacted positively to Alcian blue, dialysed iron stains and also took the Alcian blue stain in the combined aldehyde fuchsin-Alcian blue and high iron diamine-Alcian blue stains. Those of the cat gave negative results. These results suggest the presence of acidic groups in the mucosubstances secreted by the horse's duodenal glands. A suggestion is put forward on the strength of the high iron diamine-Alcian blue combined stains that the acidity is due to the presence of carboxyl groups. It is suggested that the acidity may be significant in either the cellulose metabolism or the digestion of the bacterial microflora from the stomach of herbivores.
Introduction
Received: March 29, 1975.
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
Extensive studies have been carried out on the duodenal (Brunner’s) glands of various species of mammals. Bensley [1903] established the view that pyloric glands and the duo denal glands are mucous in nature. He found that mucicarmine and mucihaemalin stained the secretory masses within the cells in all the 19 species lie studied. However, F lorf.y and H arding [1934] reported distinct species variations. They obtained good mucous reactions in the goat, pig, rabbit and guinea pig. but none at all in the cat, dog and rat. Belanger [1963], applying Hale's [H ale, 1946] and Alcian blue staining techniques, reported positive reactions in the duodenal glands of the guinea pig, domestic pig, sheep and ox, but nega tive reactions in those of the rat and man. This study was carried out to compare the reactions to Alcian blue and Hale’s dialysed iron methods between the duodenal glands of the cat and horse and then to ascertain whether the positive reactions obtained were due to carboxyl or sulphate groups present in the mucous secretion.
450
O duor -O kelo
Fig. I. Photomicrograph of the duodenal portion of the cat intestine. The goblet cells show a positive reaction. Epon-embedded block. Alcian blue, pH 2.5. x 140. Fig. 2. Photomicrograph of the duodenal portion of the horse intestine. Both the goblet cells and Brunner’s glands reacted positively giving a bluish-green colour. Paraffin-em bedded block. Alcian blue, pH. 2.5. * 250.
The duodenal samples were obtained from six cats and four horses. Small pieces, fixed in Bouin's fluid or 10% formalin, were embedded in paraffin and cut on a microtome as usual. After dewaxing and bringing the sections to water, they were stained in periodic acidSchifT (PAS) reagent after M c M anus [1946], Alcian blue after Steedman [1950], Alcian blue in different concentration of magnesium chloride after S cott and D ori.ino [1965], Alcian blue-PAS after Mowry [1963], mixed diamine solutions after S picer [1965] and aldehyde fuchsin-Alcian blue sequence after S picer and M eyer [I960], Small pieces from the cat duodenum were fixed in 2.5% giuteraldehyde, osmicated and finally embedded in Epon. Thick sections were stained in PAS solutions after N evalainen et al. [1972] without
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
Materia! and methods
Histochemistry of the duodenal glands of the cat and horse
451
Fig.3. Photomicrograph of the cat duodenum. The goblet cells gave a positive blue colour but Brunner’s glands gave the magenta colour of PAS. Epon-embedded block. Combined Alcian bluc-PAS stain, x 140. Fig.4. Photomicrograph of the horse duodenum. Both the goblet cells and Brunner's glands gave the positive bluish-purple colour. Paraffin-embedded block. Combined Alcian blue-PAS stain, x 140.
prior removal of Epon. After removal of Epon, using 20% NaOH in absolute alcohol, some slides were stained in Alcian blue solutions.
In both species the goblet cells and the duodenal glands gave PAS-positive results. While the goblet cells in both species gave positive Alcian blue and Hale’s [H ale , 1946] stains, only the duodenal glands of the horse gave positive stains to both Alcian blue and Hale's techniques (fig. 1,2). In the combined Alcian blue-PAS or dialysed iron-PAS stains the duodenal glands of the cat gave positive reactions only to PAS, while those of the horse stained a positive pale to dark-blue colour (fig. 3,4).
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
Results
452
O duor /O kelo
When the sections were stained in aldehyde fuchsin-Alcian blue sequence, the goblet cells in both cat and horse exhibited a dark-blue colour. The duodenal glands of the horse took the blue colour of Alcian blue while those of the cat did not take any colour (fig. 5,6). The mixed diamine-NaCI stain revealed positive black stains only in the goblet cells in both species. With the high iron diamine-Alcian blue sequence the golbet cells ex hibited the positive black stain, but only the duodenal glands of the horse took the Alcian blue stain: those in the cat were unstained (fig.7.8).
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Fig.5. Photomicrograph of the cat duodenum. The goblet cells are clearly positive, while Brunner's glands are not. Paraffin section. Aldehyde fuchsin. x 140. Fig.6. Photomicrograph of the horse duodenum. The goblet cells gave a bluish-purple colour, while Brunner's glands gave a greenish colour. Paraffin section. Combined aldehyde fuchsin-Alcian blue stain, x 140.
Histochemistry of the duodenal glands of the cat and horse
453
Fig. 7. Photomicrograph of the cat duodenum. The goblet cells gave a positive brownishblack colour, while Brunner's glands remained uncolourcd. Paraffin sections. Combined high iron diamine-Alcian blue stain, x 140. Fig.8. Photomicrograph of the horse duodenum. The goblet cells gave a positive brownish-black colour, while Brunner's glands took a greenish-blue colour. Paraffin section. Combined high iron diamine-Alcian blue stain, x 140.
The two types of animals used in this study belong to two different mam malian orders: the cat belonging to the order Carnivora and the horse to the order Perissodactyla. The two mammals have entirely different feeding habits. It is revealed in this histochemical study that the duodenal glands in the two mammalian species react differently to Alcian blue, dialysed iron, alde hyde fuchsin and the diamine stains. Alcian blue and dialysed iron stains are commonly used in histochemistry to identify acidic mucosubstances [P earse , 1968], while combinations of aldehyde fuchsin-Alcian blue stains
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
Discussion
454
O duor -O kelo
1965], Alcian blue, pH 1.0, and Alcian blue in various electrolyte concen trations [Scott and D orling , 1965] are generally employed in histochemistry to distinguish between sulphate (-SO.,)- and carboxyl (-COOH)- containing mucosubstances [Pearse, 1968, app. 10], The results obtained in this study show that the secretions of the duodenal glands in the horse are acidic, while those of the cat are neutral. It is further shown that the nature of acidity of the mucosubstances from the duodenal glands is different from that of the goblet cell mucosubstances. The goblet cells in both species reacted positively to aldehyde fuchsin and high iron diamine, indicating the presence of sulphate groups. When these stains were followed by Alcian blue, pH 2.5, only the duodenal glands of the horse took the blue stain, indicating the presence of carboxyl groups. Similar findings have been reported by Belanger [1963] who found that the duodenal glands of the guinea pig, domestic pig, sheep and ox, were positive to Alcian blue and dialysed iron stains while those of man and rat were negative. However, Belanger [1963] did not differentiate between sulphated and carboxylated acidic mucosubstances. Autoradiographic studies by C urran and Kennedy [1955], K ent et al. [1956], J ennings and F lorey [1956] lend support to the findings in this study. These authors reported that, whereas the goblet cells in the intestinal tract of all the animals they studied were able to incorporate sulphur, there were distinct species variations with regards to Brunner's glands. J ennings and F lorey [1956] reported that in the mouse, rat and cat there was no autoradiographic incorporation of sulphur by Brunner's glands, while there was a strong to weak radioactivity in those of the guinea pig and rabbit, respectively. C urean and K ennedy [1955] stated that the auto radiographic presence of :i5S in mouse tissues was invariably associated with mucopolysaccharides and the highest activities were in those cells which produce mucopolysaccharides, e.g. intestinal goblet cells, cartilage cells and mast cells. Spicer and M eyer [I960] have reported that both superficial and deep Brunner's glands in the guinea pig reacted positively to aldehyde fuchsin, indicating the presence of sulphated mucopolysaccharide secretions. In this study the duodenal glands of the horse did not give positive reactions with either aldehyde fuchsin or high iron diamine stains. However, it is concluded that the acidity of the mucopolysaccharide secretions in the duodenal gland of the horse may be due to the presence of carboxyl groups. The significance of the species variations in the nature of secretions of the duodenal glands is not yet clear. The acidity of the mucosubstances,
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
[Spicer and M eyer, 1950], high iron diamine-AIcian blue stain [Spicer,
Histochemistry of the duodenal glands of the cat and horse
455
which may be due to carboxyl groups in the horse, but may be due to both carboxyl and sulphate groups in the guinea pig, might well be correlated to the differences in the feeding habits of the various species. The findings in this study and in previous ones seem to show that those animals which feed on cellulose-containing substances (herbivores) secrete acidic mucosubstances from their duodenal glands. These groups of animals also tend to have much more extensive development of the duodenal glands than those animals in which the duodenal secretions are neutral. It is finally postulated that the acidic mucosubstances secreted by the duodenal glands of herbivores may play an important role either in end-products of cellulose metabolism or in the metabolism of microfloral (bacterial) proteins especially in the case of ruminants. This postulation will require more histochemical and biochemical analysis. A cknowledgements This work was made possible through the Swiss Federal Government scholarship. I wish to express my sincere gratitude to Prof. Dr .W. Mosimann who offered me the facil ities in his Institute. My thanks go to Dr. R. Leiser for useful discussions and constant encouragement. I am greatly indected to Mrs. H .K opf, Miss B.H ermansson, Miss P. ROedi, Mr. U .I ff and Mr. E. G asser for their invaluable technical assistance. I should also like to thank Miss A. Ikua of the University of Nairobi who agreed to type the final script.
References Belanger, L. F .: Comparison between different histochemical and histophysical techniques
as applied to mucus-secreting cells. Ann. N.Y. Acad. Sci. 106: 364-378 (1963). Bensley. R.R.: Decennial Pubis Univ. Chicago 10: 279(1903). C urran . R.C. and K ennedy, J.S.: The distribution of the sulfated mucopolysaccharides
in the mouse. J. Path. Bact. 70: 449-457 (1955). F lorey, H.W . and H arding , H .E .: Further observations on the secretion of Brunner's
glands. J. Path. Bact. 39: 255-276 (1934). H ale, C. W.: Histochemical demonstration of acid polysaccharides in animal tissues.
Nature, Lond. 157: 802(1946). J ennings, M.A. and F lorey, H.W .: Autoradiographic observations on the mucous cells
of the stomach and intestines. Q. Jl exp. Physiol. 41: 131-151 (1956). M c M anus, J.F.A .: Histological demonstration of mucin after periodic acid. Nature,
Lond. 158: 202 (1946). M owry , R. W.: The special value of methods that colour both acidic and vicinal hydroxyl
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
groups in the histochemical iron stain, the use of Alcian blue 8 G x and their combi nations with the periodic acid-Schiff reaction, Ann. N.Y. Acad. Sci. 106:402-423 (1963).
456
O duor /O kelo
N evalainen, T.J.; Laitio, M., and Lindcren , I.: Periodic acid-SehilT (PAS) staining of
Epon-embedded tissue for light microscopy. Acta histocheni. 42: 230-233 (1972). P earse, A .G .E.: Histochemistry: theoretical and applied; 3rd ed., vol. 1 (Churchill.
London 1968). Scott . J.E . and D orlinci, J.: Differential staining of acid glycosaminoglycans (muco
polysaccharides) by Alcian blue in salt solutions. Histochemie 5: 221-233 (1965). S picer, S.S.: Diamine methods for differentiating mucosubstances histochemically. J.
Histocheni. Cytochcm. 13: 211 234 (1965). S picer, S.S. and M eyer. D. B.: Histochcmical differentiation of acid mucopolysaccharides
D. O duor-O kelo, The University of Nairobi, Department of Veterinary Anatomy, PO Box 30197, Nairobi (Kenya)
Downloaded by: UCL 144.82.238.225 - 2/6/2018 8:45:56 PM
by means of combined aldehyde fuchsin-Alcian blue staining. Am. J. clin. Path. 33: 453-460(1960). S teedman, H .F.: Alcian blue 8 GS: a new stain for mucins. Q. Jl microsc. Sci. 91: 447449(1950).
