Histochemistry 48, 153 - 159 (1976)
Histochemistry 9 by Springer-Verlag 1976
An Alcohol-Soluble Schiff's Reagent: A Histochemical Application of the Complex between Schiff's Reagent and Phosphotungstic Acid K.S. Bedi and Richard W. Horobin Department of Human Biology and Anatomy, The University of Sheffield, 3 Ctarkehouse Road, Sheffield $10 2TN, U.K.
Summary. A schedule for staining partially hydrated PAS-positive structures using non-aqueous solutions has been devised. Tissues are dewaxed, taken down to 70% alcohol, oxidised for 10 rain in a 1% w/v alcoholic solution of periodic acid, treated with an alcoholic solution of phosphotungstic acidSchiff reagent complex (PTA-Schiff reagent) for 25 min, washed in alcohol, cleared in xylene and mounted in a synthetic medium. The PTA-Schiff reagent complex prepared from de Tomasi Schiff reagent by precipitation with PTA may be stored in the deep freeze for many months and dissolved freshly in alcohol for use. The PTA-Schiff reagent used as above allows staining of highly water soluble materials such as dextran. From blocking and digestion studies the mode of action seems similar to de Tomasi Schiff reagent. The partial hydration of the tissues prior to reaction was found to be essential for effective staining.
Introduction Certain polysaccharides, mucosubstances and fatty acids are extremely water soluble and consequently their histochemical demonstration by the standard periodic acid-Schiff (PAS) procedure using aqueous solutions of periodic acid and of Schiff's reagent has proved difficult. In efforts to circumvent this problem the use of an alcoholic solution of periodic acid and aqueous-alcoholic solutions of Schiff reagent have been recommended (Schibsted, 1932; Staple, 1957; Mowry et al., 1952). Such ~ alcoholic Schiff reagents" have proved of limited usefulness, apparently due to the low solubility of the parent dye. To overcome this, Kasten (1959, 1960) suggested the use of alcohol soluble Schiff-type reagents, prepared from dyes such as typogen brown and magdala red, for the demonstration of water soluble polysaccharides. None of these procedures appear to have achieved general acceptance. A recent observation (Scott, 1973) that aqueous phosphotungstic acid (PTA) added to Schiff reagent "produces a copious white microcrystalline precipitate"
154
K.S. Bedi and R.W. Horobin
of a PTA-Schiff reagent complex suggested another approach to this problem. Consequently we have investigated the use of an alcoholic solution of this PTA-Schiff reagent complex as a new non-aqueous Schiff reagent.
Materials and Methods Preparation of Reagents Pilot experiments had previously shown that the PTA-Schiff complex was reasonably soluble in absolute alcohol giving a pink solution. This solution reacted with formaldehyde producing a purple colour. A standard method, detailed below, for the preparation of this alcoholic PTA-Schiff reagent was developed empirically. A known volume of fresh de Tomasi Schiff reagent (Pearse, 1968, p. 647) prepared using basic fuchsin (batch no. 1941 supplied by Raymond A. Lamb) was added to an equal volume of 1% w/v aqueous PTA (B.D.H. Ltd.). The resulting white precipitate of PTA-Schiff complex was centrifuged down and the supernate discarded. The precipitate was then redissolved in the same volume of absolute alcohol as was originally used to prepare the PTA-Schiff complex. The staining properties of this alcoholic PTA-Schiff reagent were examined.
Fixatives and Tissues Various rat tissues were fixed in an variety of fixatives and embedded in paraffin wax. 6 gm sections were cut on a microtome and mounted on clean slides. The PTA-Schiff reagent was also tested on a model system, consisting of slides which had been streaked with a solution of dextran and then allowed to air dry.
Staining Procedures The staining schedules and reagents were variants on the usual PAS procedure, thus:
Variant a 1. Sections were dewaxed in xylene for five minutes, 2. brought through a series of alcohols to water, 3. treated with 1% w/v aqueous periodic acid for ten minutes, 4. washed for 2-3 rain in running tap water, 5. treated with either ethanolic PTA-Schiff reagent or de Tomasi Schiff reagent for varying periods of time up to six hours, 6. washed in running tap water for twenty minutes, 7. dehydrated, cleared in xylene and mounted in Polymount (Stayne Laboratories).
Variant b 1. 2. 3. 4. 5. 6. 7.
Sections were dewaxed in xylene for five minutes, brought through a series of alcohol to water, treated with 1% w/v alcoholic periodic acid for periods of time up to sixty minutes, washed for 2-3 rain in absolute alcohol, treated with either ethanolic PTA-Schiff reagent or de Tomasi Schiff reagent, washed in alcohol for twenty minutes, cleared in xylene and mounted in Polymount.
An Alcohol-Soluble Schiff's Reagent
155
Variant c As for variant (b) except step 2 at which point the sections were only brought down either to 70% or absolute ethanol.
Blocking and Extraction After sections had been oxidised with periodic acid as in variant (a) they were further treated with either fresh saliva for thirty minutes at 37~ or with an aldehyde blocking agent consisting of a mixture of aniline and acetic acid (10:90) for thirty minutes at room temperature (Pearse, 1968). Staining was then carried out in either the alcoholic PTA-Schiff reagent or the de Tomasi Schiff reagent as before.
Feutgen Procedure Tissue sections were hydrolysed in 5N HC1 at 25~ for sixty minutes, washed in running tap water, rinsed in distilled water and then stained in either alcoholic PTA-Schiff reagent or de Tomasi Schiff reagent for forty minutes. Sections were then rinsed in distilled water, dehydrated in a series of alcohols, cleared in xylene and mounted in Polymount.
Results and Discussion
Observations after Staining Sections by the PAS Procedure When using the variant (a) staining procedure, results achieved with PTA Schiff were comparable with control slides which had been stained with de Tomasi Schiff reagent (see Figs. 1 and 2). With both Schiff reagents the staining patterns and intensities were related to the fixative employed and the duration of staining as well as the particular tissue being stained. Thirty minutes staining in the alcoholic PTA-Schiff reagent was sufficient to give reasonable staining in most cases. When using the variant (b) staining procedure the results obtained were similar to those with variant (a). The staining patterns and intensities appeared to be unaffected, both in the control and the experimental slides, by the use of the alcoholic periodic acid in place of the normal aqueous solution, see Figures 3 and 4. When using the variant (c) staining procedure sections taken down to absolute alcohol only and stained in the alcoholic solution of the PTA-Schiff reagent showed no signs of staining whatsoever even when the staining time was increased up to six hours. Staining times longer than this presented problems because of the lack of stability of the alcoholic PTA-Schiff reagent. The control sections treated in a similar way but stained with the aqueous de Tomasi Schiff reagent also showed very little staining. Use of schedule (c), but allowing partial hydration by taking the sections down to 70% ethanol, yielded reasonable staining both for those sections stained in the alcoholic PTA-Schiff reagent or in de Tomasi Schiff reagent (Fig. 5). Thus it appears that some hydration is necessary for staining with the PAS procedure whichever variant is used. The general importance of this phenomenon
156
K.S. Bedi and R.W. Horobin
An Alcohol-Soluble Schiff's Reagent
157
Figs. 1 and 2. Neutral formalin fixed rat kidney and gut tissue stained by the PAS procedure. Sections were dewaxed in xylene, taken through a series of alcohols to water, and then treated with aqueous periodic acid before staining either in the alcoholic PTA-Schiff reagent (a) or de Tomasi Schiff reagent, (b). x 300 Figs. 3 and 4. Gendre fixed rat trachea and gut tissue stained by the PAS procedure. Sections were dewaxed in xylene, taken through a series of alcohols to water, and then treated with alcoholic periodic acid before staining either in the alcoholic PTA-Schiff reagent (a) or the de Tomasi Schiff reagent, (b). • 300 Fig. 5. Neutral formalin fixed rat trachea stained by the PAS procedure. Sections were dewaxed in xylene, taken down to 70% alcohol and then treated with alcoholic periodic acid, before staining either in the alcoholic PTA-Schiff reagent (a) or the de Tomasi Schiff reagent, (b). x 300
158
K.S. Bedi and R.W. Horobin
to other simpler one step staining systems was also investigated (to be reported elsewhere). Analogous observations in other systems have been reported, e.g. in the Gram stain (Tucker and Bartholomew, 1962). The potential usefulness of the alcoholic PTA-Schiff reagent in a completely non-aqueous staining schedule (variant c) was demonstrated on a model system comprising test slides streaked with dextran. Staining of these test slides using de Tomasi Schiff reagent proved unsuccessful, apparently due to the extreme solubility of the dextran. Staining of the test slides was possible when the alcoholic PTA-Schiff reagent was used.
Mode of Action of Alcoholic PTA-Schiff Reagent The alcoholic PTA-Schiff reagent appears to act in a manner similar to the more usual de Tomasi solution. This is indicated by the experiments investigating the effects of blocking and extraction procedures on the staining patterns and intensities using both the alcoholic PTA-Schiff reagent and de Tomasi Schiff reagent. The staining by both the reagents was eliminated by preliminary treatment with aniline-acetic acid, and greatly reduced by the enzymic digestion.
Staining by the Feulgen Procedure Control sections stained with de Tomasi Schiff reagent showed the characteristic nuclear staining associated with the Feulgen procedure. However experimental sections, stained with alcoholic PTA-Schiff reagent produced no staining at all.
Properties of the Alcoholic PTA-Schiff Reagent The alcoholic solution of the PTA-Schiff reagent was unstable both at room temperature and at 4~ turning from a pink to a deep purple colour within a few hours. However the PTA-Schiff precipitate, on drying and freezing at 0~ could be successfully stored for several months after which time it still retained its property of dissolving in absolute alcohol to yield a solution which reacted with formaldehyde. This observation may provide an answer to the quest for a stable solid form of Schiff reagent. Acknowledgements. We would like to thank Professor R. Barer for encouragement and advice. K.S.B. was in receipt of the John Stokes Research Fellowship.
References Kasten, F.H.: Schiff-type reagents in cytochemistry. I. Theoretical and practical considerations. Histochemie 1, 466-509 (1959)
An Alcohol-Soluble Schiff's Reagent
159
Kasten, F.H.: The chemistry of Schiff's reagent. Int. Rev. Cytol. 10, 1-100 (G.H. Bourne and J.F. Danielli, ed.). New York: Academic Press 1960 Mowry, R.W., Longley, J.B., Millican, R.C. : Histochemical demonstration of intravenously injected dextran in kidney and liver of the mouse. J. Lab. clin. Med. 39, 211-217 (1952) Pearse, A.G.E. : I-Iistochemistry theoretical and applied, 3rd ed., Vol. 1. London: J.&A. Churchill Ltd. 1968 Schibsted, H.: New test for fat aldehydes resulting from oxidation of fats and oils. Ind. Eng. Chem. Anal. Ed. 4, 204-208 (1932) Scott, J.E.: PTA 'Schiff reactive' but not a glycol reagent. J. Histochem. Cytochem. 21, 1084 (1973) Staple, P,H.: The effect of boric acid on the reaction of lead tetra acetate with polysaccharides in films and tissue sections. J. Histochem. Cytochem. 5, 472-488 (I957) Tucker, F.L., Bartholomew, J.W. : Variations in the Gram staining; results caused by air moisture. Stain Technol. 37, 157-160 (1962)
Received Februaryl4, 1976
Histochemistry 9 by Springer-Verlag 1976
An Alcohol-Soluble Schiff's Reagent: A Histochemical Application of the Complex between Schiff's Reagent and Phosphotungstic Acid K.S. Bedi and Richard W. Horobin Department of Human Biology and Anatomy, The University of Sheffield, 3 Ctarkehouse Road, Sheffield $10 2TN, U.K.
Summary. A schedule for staining partially hydrated PAS-positive structures using non-aqueous solutions has been devised. Tissues are dewaxed, taken down to 70% alcohol, oxidised for 10 rain in a 1% w/v alcoholic solution of periodic acid, treated with an alcoholic solution of phosphotungstic acidSchiff reagent complex (PTA-Schiff reagent) for 25 min, washed in alcohol, cleared in xylene and mounted in a synthetic medium. The PTA-Schiff reagent complex prepared from de Tomasi Schiff reagent by precipitation with PTA may be stored in the deep freeze for many months and dissolved freshly in alcohol for use. The PTA-Schiff reagent used as above allows staining of highly water soluble materials such as dextran. From blocking and digestion studies the mode of action seems similar to de Tomasi Schiff reagent. The partial hydration of the tissues prior to reaction was found to be essential for effective staining.
Introduction Certain polysaccharides, mucosubstances and fatty acids are extremely water soluble and consequently their histochemical demonstration by the standard periodic acid-Schiff (PAS) procedure using aqueous solutions of periodic acid and of Schiff's reagent has proved difficult. In efforts to circumvent this problem the use of an alcoholic solution of periodic acid and aqueous-alcoholic solutions of Schiff reagent have been recommended (Schibsted, 1932; Staple, 1957; Mowry et al., 1952). Such ~ alcoholic Schiff reagents" have proved of limited usefulness, apparently due to the low solubility of the parent dye. To overcome this, Kasten (1959, 1960) suggested the use of alcohol soluble Schiff-type reagents, prepared from dyes such as typogen brown and magdala red, for the demonstration of water soluble polysaccharides. None of these procedures appear to have achieved general acceptance. A recent observation (Scott, 1973) that aqueous phosphotungstic acid (PTA) added to Schiff reagent "produces a copious white microcrystalline precipitate"
154
K.S. Bedi and R.W. Horobin
of a PTA-Schiff reagent complex suggested another approach to this problem. Consequently we have investigated the use of an alcoholic solution of this PTA-Schiff reagent complex as a new non-aqueous Schiff reagent.
Materials and Methods Preparation of Reagents Pilot experiments had previously shown that the PTA-Schiff complex was reasonably soluble in absolute alcohol giving a pink solution. This solution reacted with formaldehyde producing a purple colour. A standard method, detailed below, for the preparation of this alcoholic PTA-Schiff reagent was developed empirically. A known volume of fresh de Tomasi Schiff reagent (Pearse, 1968, p. 647) prepared using basic fuchsin (batch no. 1941 supplied by Raymond A. Lamb) was added to an equal volume of 1% w/v aqueous PTA (B.D.H. Ltd.). The resulting white precipitate of PTA-Schiff complex was centrifuged down and the supernate discarded. The precipitate was then redissolved in the same volume of absolute alcohol as was originally used to prepare the PTA-Schiff complex. The staining properties of this alcoholic PTA-Schiff reagent were examined.
Fixatives and Tissues Various rat tissues were fixed in an variety of fixatives and embedded in paraffin wax. 6 gm sections were cut on a microtome and mounted on clean slides. The PTA-Schiff reagent was also tested on a model system, consisting of slides which had been streaked with a solution of dextran and then allowed to air dry.
Staining Procedures The staining schedules and reagents were variants on the usual PAS procedure, thus:
Variant a 1. Sections were dewaxed in xylene for five minutes, 2. brought through a series of alcohols to water, 3. treated with 1% w/v aqueous periodic acid for ten minutes, 4. washed for 2-3 rain in running tap water, 5. treated with either ethanolic PTA-Schiff reagent or de Tomasi Schiff reagent for varying periods of time up to six hours, 6. washed in running tap water for twenty minutes, 7. dehydrated, cleared in xylene and mounted in Polymount (Stayne Laboratories).
Variant b 1. 2. 3. 4. 5. 6. 7.
Sections were dewaxed in xylene for five minutes, brought through a series of alcohol to water, treated with 1% w/v alcoholic periodic acid for periods of time up to sixty minutes, washed for 2-3 rain in absolute alcohol, treated with either ethanolic PTA-Schiff reagent or de Tomasi Schiff reagent, washed in alcohol for twenty minutes, cleared in xylene and mounted in Polymount.
An Alcohol-Soluble Schiff's Reagent
155
Variant c As for variant (b) except step 2 at which point the sections were only brought down either to 70% or absolute ethanol.
Blocking and Extraction After sections had been oxidised with periodic acid as in variant (a) they were further treated with either fresh saliva for thirty minutes at 37~ or with an aldehyde blocking agent consisting of a mixture of aniline and acetic acid (10:90) for thirty minutes at room temperature (Pearse, 1968). Staining was then carried out in either the alcoholic PTA-Schiff reagent or the de Tomasi Schiff reagent as before.
Feutgen Procedure Tissue sections were hydrolysed in 5N HC1 at 25~ for sixty minutes, washed in running tap water, rinsed in distilled water and then stained in either alcoholic PTA-Schiff reagent or de Tomasi Schiff reagent for forty minutes. Sections were then rinsed in distilled water, dehydrated in a series of alcohols, cleared in xylene and mounted in Polymount.
Results and Discussion
Observations after Staining Sections by the PAS Procedure When using the variant (a) staining procedure, results achieved with PTA Schiff were comparable with control slides which had been stained with de Tomasi Schiff reagent (see Figs. 1 and 2). With both Schiff reagents the staining patterns and intensities were related to the fixative employed and the duration of staining as well as the particular tissue being stained. Thirty minutes staining in the alcoholic PTA-Schiff reagent was sufficient to give reasonable staining in most cases. When using the variant (b) staining procedure the results obtained were similar to those with variant (a). The staining patterns and intensities appeared to be unaffected, both in the control and the experimental slides, by the use of the alcoholic periodic acid in place of the normal aqueous solution, see Figures 3 and 4. When using the variant (c) staining procedure sections taken down to absolute alcohol only and stained in the alcoholic solution of the PTA-Schiff reagent showed no signs of staining whatsoever even when the staining time was increased up to six hours. Staining times longer than this presented problems because of the lack of stability of the alcoholic PTA-Schiff reagent. The control sections treated in a similar way but stained with the aqueous de Tomasi Schiff reagent also showed very little staining. Use of schedule (c), but allowing partial hydration by taking the sections down to 70% ethanol, yielded reasonable staining both for those sections stained in the alcoholic PTA-Schiff reagent or in de Tomasi Schiff reagent (Fig. 5). Thus it appears that some hydration is necessary for staining with the PAS procedure whichever variant is used. The general importance of this phenomenon
156
K.S. Bedi and R.W. Horobin
An Alcohol-Soluble Schiff's Reagent
157
Figs. 1 and 2. Neutral formalin fixed rat kidney and gut tissue stained by the PAS procedure. Sections were dewaxed in xylene, taken through a series of alcohols to water, and then treated with aqueous periodic acid before staining either in the alcoholic PTA-Schiff reagent (a) or de Tomasi Schiff reagent, (b). x 300 Figs. 3 and 4. Gendre fixed rat trachea and gut tissue stained by the PAS procedure. Sections were dewaxed in xylene, taken through a series of alcohols to water, and then treated with alcoholic periodic acid before staining either in the alcoholic PTA-Schiff reagent (a) or the de Tomasi Schiff reagent, (b). • 300 Fig. 5. Neutral formalin fixed rat trachea stained by the PAS procedure. Sections were dewaxed in xylene, taken down to 70% alcohol and then treated with alcoholic periodic acid, before staining either in the alcoholic PTA-Schiff reagent (a) or the de Tomasi Schiff reagent, (b). x 300
158
K.S. Bedi and R.W. Horobin
to other simpler one step staining systems was also investigated (to be reported elsewhere). Analogous observations in other systems have been reported, e.g. in the Gram stain (Tucker and Bartholomew, 1962). The potential usefulness of the alcoholic PTA-Schiff reagent in a completely non-aqueous staining schedule (variant c) was demonstrated on a model system comprising test slides streaked with dextran. Staining of these test slides using de Tomasi Schiff reagent proved unsuccessful, apparently due to the extreme solubility of the dextran. Staining of the test slides was possible when the alcoholic PTA-Schiff reagent was used.
Mode of Action of Alcoholic PTA-Schiff Reagent The alcoholic PTA-Schiff reagent appears to act in a manner similar to the more usual de Tomasi solution. This is indicated by the experiments investigating the effects of blocking and extraction procedures on the staining patterns and intensities using both the alcoholic PTA-Schiff reagent and de Tomasi Schiff reagent. The staining by both the reagents was eliminated by preliminary treatment with aniline-acetic acid, and greatly reduced by the enzymic digestion.
Staining by the Feulgen Procedure Control sections stained with de Tomasi Schiff reagent showed the characteristic nuclear staining associated with the Feulgen procedure. However experimental sections, stained with alcoholic PTA-Schiff reagent produced no staining at all.
Properties of the Alcoholic PTA-Schiff Reagent The alcoholic solution of the PTA-Schiff reagent was unstable both at room temperature and at 4~ turning from a pink to a deep purple colour within a few hours. However the PTA-Schiff precipitate, on drying and freezing at 0~ could be successfully stored for several months after which time it still retained its property of dissolving in absolute alcohol to yield a solution which reacted with formaldehyde. This observation may provide an answer to the quest for a stable solid form of Schiff reagent. Acknowledgements. We would like to thank Professor R. Barer for encouragement and advice. K.S.B. was in receipt of the John Stokes Research Fellowship.
References Kasten, F.H.: Schiff-type reagents in cytochemistry. I. Theoretical and practical considerations. Histochemie 1, 466-509 (1959)
An Alcohol-Soluble Schiff's Reagent
159
Kasten, F.H.: The chemistry of Schiff's reagent. Int. Rev. Cytol. 10, 1-100 (G.H. Bourne and J.F. Danielli, ed.). New York: Academic Press 1960 Mowry, R.W., Longley, J.B., Millican, R.C. : Histochemical demonstration of intravenously injected dextran in kidney and liver of the mouse. J. Lab. clin. Med. 39, 211-217 (1952) Pearse, A.G.E. : I-Iistochemistry theoretical and applied, 3rd ed., Vol. 1. London: J.&A. Churchill Ltd. 1968 Schibsted, H.: New test for fat aldehydes resulting from oxidation of fats and oils. Ind. Eng. Chem. Anal. Ed. 4, 204-208 (1932) Scott, J.E.: PTA 'Schiff reactive' but not a glycol reagent. J. Histochem. Cytochem. 21, 1084 (1973) Staple, P,H.: The effect of boric acid on the reaction of lead tetra acetate with polysaccharides in films and tissue sections. J. Histochem. Cytochem. 5, 472-488 (I957) Tucker, F.L., Bartholomew, J.W. : Variations in the Gram staining; results caused by air moisture. Stain Technol. 37, 157-160 (1962)
Received Februaryl4, 1976
