Skip to main content
search
Category

Aldehydes

Pseudo-Periodic Acid Schiff

By Aldehydes, Protocols, Pseudo-Schiff Reaction, Schiff's Reagent Reactions, Stain Target, Stain Type
Protocol

Pseudo-Periodic Acid Schiff

10
steps
2
materials
print

The terms Pseudo-Schiff and Pseudo-PAS refer to the use of dyes other than pararosanilin or basic fuchsin to make a Schiff type solution and use it for demonstrating carbohydrates. While an interesting exercise, it has little practical use except for the occasional demonstration of fungi with a fluorescent pseudo-Schiff solution made with acriflavine. The following dyes, among others, have been suggested.

DyeCI NumberColor
Acid fuchsin42685violet
Acriflavine46000yellow
Azure A52005blue
Azure C52005blue
Crystal violet42555blue-violet
Methyl violet42535violet
Methylene blue52015blue
Safranin O50240red
Thionin52005blue
Toluidine blue52040blue

Materials

  • 1% hydrochloric acid in 70% ethanol
  • A suitable counterstain

Preparation of Pseudo-Schiff Solution

Prepare a solution according to the instructions for acriflavine Schiff reagent, or another formula, if preferred. Note that they will likely not become colourless, so leave for 48 hours, then filter the solution and store refrigerated.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are usually satisfactory, although glutaraldehyde should be avoided.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into periodic acid for 10 minutes.
  3. Wash with running water.
  4. Rinse with distilled water.
  5. Place in pseudo-Schiff’s reagent for 10-30 minutes.
  6. Rinse with distilled water.
  7. Place in acid ethanol for 5 minutes.
  8. Wash well with tap water for about 10 minutes.
  9. Counterstain with a suitable contrasting nuclear stain.
  10. Dehydrate with ethanol, clear with xylene and coverslip using a resinous medium.

Expected Results

  • Oxidisable carbohydrates – coloured according to the dye used
  • Nuclei – as stained

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Culling C.F.A., (1963)
    Handbook of histopathological and histochemical techniques Ed. 2
    Butterworth, London, UK.
March 30, 2023
Love0

Fluorescent Nucleal Reaction for DNA

By Aldehydes, Nucleal Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type
Protocol

Fluorescent Nucleal Reaction

for DNA

9
steps
3
materials
print

Materials

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Many other fixatives are satisfactory. Fixatives containing strong acids should be avoided as this method depends on the acid hydrolysis of DNA, and acids in some fixatives may pre-hydrolyse the tissue (picric acid in Bouin’s aqueous formal-picric-acetic mixture for example).

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse briefly with cold 1 N hydrochloric acid.
  3. Place into prewarmed hydrochloric acid for the appropriate time at 60°C.
  4. Rinse briefly with cold 1 N hydrochloric acid.
  5. Rinse briefly with distilled water.
  6. Place into Acriflavine Schiff’s reagent for 30-60 minutes at room temperature.
  7. Place into 1% acid alcohol, 2 changes for about 5 minutes each.
  8. Wash well with water.
  9. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Using a BG 12 exciter filter, and OG 4 (yellow) and/or OG5 (orange) barrier filter, DNA fluoresces yellow.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Culling, C F A, Allison, R T, Barr, W T, (1985).
    Cellular pathology technique., Ed. 4, p. 189.
    Butterworths, London, England.
March 30, 2023
Love0

Hotchkiss’ Alcoholic Periodic Acid Schiff

By Aldehydes, Periodic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type
Protocol

Hotchkiss' Alcoholic Periodic Acid Schiff

13
steps
10
materials
print

It was initially thought that the periodic acid Schiff reaction could result in less glycogen being demonstrated than was actually present because it might dissolve in aqueous reagents. It is now known this is not a concern. Hotchkiss recommended an alcoholic method to ensure it did not take place. This method is now redundant.

Materials

  • Schiff’s reagent
  • Mayer’s hemalum
  • Alcoholic periodic acid
    MaterialAmount
    Periodic acid0.8g
    Sodium acetate buffer 0.2M10mL
    Ethanol, absolute70mL
    Distilled water20mL
  • Acid reducing rinse
    MaterialAmount
    Potassium iodide2g
    Sodium thiosulphate2g
    Ethanol absolute60mL
    Hydrochloric acid N/12mL
    Distilled water40mL

Tissue Sample

Presumably an alcoholic fixative should be required if glycogen were to dissolve in aqueous solutions. However, 5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are usually satisfactory, although glutaraldehyde should be avoided.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into periodic acid for 10 minutes.
  3. Rinse with 70% ethanol.
  4. Place in acid reducing rinse for 1 minute.
  5. Rinse with 70% ethanol.
  6. Wash with running water to remove ethanol.
  7. Rinse with distilled water.
  8. Place in Schiff’s reagent for 10-30 minutes.
  9. Wash off with distilled water.
  10. Wash well with tap water for about 10 minutes.
  11. Counterstain with Mayer’s hemalum for 1 minutes.
  12. Wash well with tap water until hemalum is blued.
  13. Dehydrate with ethanol, clear with xylene, and coverslip using a resinous medium.

Expected Results

  • Oxidisable carbohydrates – red
  • Nuclei – blue

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Culling C.F.A., (1963)
    Handbook of histopathological and histochemical techniques Ed. 2
    Butterworth, London, UK.
March 30, 2023
Love0

Periodic Acid Schiff Diastase

By Aldehydes, Periodic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type
Protocol

Periodic Acid Schiff Diastase

15
steps
4
materials
print

The periodic acid Schiff reaction (PAS) is used to demonstrate the presence of 1-2-glycols, including glycogen. Sometimes it is necessary for the glycogen to be removed so that a clearer picture of non-glycogen carbohydrates may be seen. Glycogen may be removed with amylase (diastase).

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are usually satisfactory, although glutaraldehyde should be avoided.

Protocol

  1. Two consecutive test and 2 consecutive control sections.
  2. Bring sections to water via xylene and ethanol.
  3. Place one test section and one known positive section into water.
  4. Place the other test section and the other known positive in the amylase solution for 30-60 minutes at 35-45°C
  5. Wash both digested sections with tap water
  6. Celloidinise the sections if desired.
  7. Place into periodic acid for 10-30 minutes.
  8. Rinse well with tap water.
  9. Rinse with distilled water.
  10. Place in Schiff’s reagent for 10-30 minutes.
  11. Wash off with distilled water.
  12. Wash well with tap water for about 10 minutes.
  13. Counterstain with Mayer’s hemalum for 2 minutes.
  14. Wash well with tap water until hemalum is blued.
  15. Dehydrate with ethanol, clear with xylene and coverslip using a resinous medium.

Expected Results

  • Glycogen – red in the undigested section, absent from the digested section.
  • Oxidisable carbohydrates – red
  • Nuclei – blue

Notes

  • If the intent is to remove glycogen rather than identify it, a more convenient approach in a routine service laboratory is to dissolve the amount of α-amylase that would cover a 1 cm circle about 1 mm deep in 15 mL (1 test tube) of distilled water. Shake for a few minutes, then filter onto the sections and digest at room temperature. Glycogen is usually removed within 30 minutes to 1 hour. Wash well and continue with a regular PAS (step 7).
  • Please note that an older variation of the procedure in Note 1 was to collect saliva and use that for the digestion. This is strongly deprecated. Human body fluids may contain transmissible bacterial and viral contaminants, so anyone handling such a slide will be at risk.
  • Hog α-amylase is recommended as being consistent and effective. Purchase a product with a high α-amylase activity.
  • Glutaraldehyde fixation leaves free aldehyde groups attached to tissues, which causes an overall positive reaction. These groups may be stopped from reacting with an appropriate procedure such as the aniline-acetic aldehyde block.
  • The tap water wash at step 7 is necessary to develop the red color. Within limits, the longer the wash the darker the color.
  • Originally, it was recommended that the Schiff’s reagent be washed off with dilute sulfurous acid (the sulfite rinses). Since water recolors Schiff’s reagent, it was believed that a water wash could lead to false positive results. It is now known this is not the case, provided the Schiff’s reagent is removed quickly and the sections do not stay in water contaminated with it for extended periods.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

March 30, 2023
Love0

Gridley’s Stain for Fungi

By Aldehydes, Chromic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type
Protocol

Gridley's Stain

for Fungi

15
steps
7
materials
print

Materials

  • Schiff’s reagent
  • Aldehyde fuchsin
  • Chromic acid
    MaterialAmount
    Chromium trioxide5g
    Distilled water500mL
  • Bleach
    MaterialAmount
    Sodium metabisulfite5g
    Distilled water500mL
  • Metanil yellow
    MaterialAmount
    Metanil yellow1g
    Distilled water400mL
    Acetic acid, glacial2drops

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in chromic acid for 1 hour.
  3. Wash well with tap water.
  4. Treat with the metabisulfite bleach for 1 minute.
  5. Wash well with tap water.
  6. Rinse with distilled water.
  7. Place in Schiff’s reagent for 20 minutes.
  8. Wash well with tap water.
  9. Rinse with 70% ethanol.
  10. Place in aldehyde fuchsin 30 minutes.
  11. Rinse off excess with 95% ethanol.
  12. Wash well with tap water.
  13. Counterstain with metanil yellow for 1 minute.
  14. Rinse well with distilled water.
  15. Dehydrate, clear and mount in a resinous medium.

Expected Results

  • Fungi  –  purple
  • Background  –  yellow

Notes

  • At step 2, a 10% solution of chromic acid applied for 10 minutes will give similar results.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
  2. Humason, G. L., (1967)
    Animal tissue techniques, Ed. 2
    W. H. Freeman and Company, San Francisco, Ca, USA
March 30, 2023
Love0

Feulgen Nucleal Reaction for DNA

By Aldehydes, Nucleal Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type
Protocol

Feulgen Nucleal Reaction

for DNA

10
steps
3
materials
print

Materials

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Many other fixatives are satisfactory. Fixatives containing strong acids should be avoided as this method depends on the acid hydrolysis of DNA, and acids in some fixatives may pre-hydrolyse the tissue (picric acid in Bouin’s aqueous formal-picric-acetic mixture, for example).

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse briefly with cold 1N hydrochloric acid.
  3. Place into prewarmed hydrochloric acid for the appropriate time at 60°C.
  4. Rinse briefly with cold 1N hydrochloric acid.
  5. Rinse briefly with distilled water.
  6. Place into Schiff’s reagent for 30-60 minutes at room temperature.
  7. Give three sulphite rinses of about 1 minute each.
  8. Wash well with water.
  9. Optionally, counterstain with light green for 1 minute.
  10. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • DNA  –  red
  • Background  –  green

Notes

  • The appropriate time in hydrochloric acid varies depending on the fixative. The times given below are in minutes, but should be considered a guide only. Trials should be conducted to determine the optimum.
    FixativeTime
    BouinDo not use
    Carnoy8
    Champy25
    Flemming16
    Formalin (NBF)10
    Formal sublimate8
    Helly8
    SuSa18
    Zenker5
  • A modification of this reaction uses more concentrated hydrochloric acid at room temperature. At step 3, place into 5N hydrochloric acid at room temperature for the appropriate time. Then continue on with step 4. This variant is considered to produce darker staining and a smaller loss of DNA.
    FixativeTime
    Alcoholic fixatives20 minutes to 2 hours
    Formalin containing fixatives35 minutes to 4 hours
    Formalin vapour2 to 8 hours
  • Sulphite rinses are now considered unnecessary. They were used originally in the belief that placing directly into water would recolour the Schiff’s reagent and give false positive staining. It is now known that this is not so, providing the Schiff’s reagent is completely washed off. Sulphite rinses consist of:
    MaterialAmount
    Potassium metabisulphite, 10% aqu.5mL
    Hydrochloric acid, 1N95mL

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Pearse, A. G. E., (1968, 1972)
    Histochemistry: Theoretical and Applied, Ed. 3
    Churchill Livingstone, Edinburgh, London, UK
March 30, 2023
Love0

Periodic Acid Schiff Reaction

By Aldehydes, Periodic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type

Protocol

Periodic Acid Schiff Reaction

10
steps
3
materials
print

The periodic acid Schiff reaction (PAS) is used to demonstrate the presence of 1-2-glycols, and is consequently an important method in the histochemistry of carbohydrates and the histological demonstration of many structures.

Expected Results

Periodic acid Schiff staining in liver biopsy of glycogen storage disorder, 20X magnification

periodic-acid-schiff-staining-liver-biopsy-glycogen-storage-disorder-600x400

Figure 1. Periodic Acid Schiff Staining in Liver Biopsy of Glycogen Storage Disorder

Positive periodic acid Schiff (PAS) staining (dark purple) due to intracytoplasmic accumulation of glycogen in a liver biopsy of glycogen storage disorder shows uniformly distended hepatocytes with clear to pale eosinophilic cytoplasm. 20X magnification. Liver in glycogen storage disease, PAS stain by Department of Pathology, Calicut Medical College is licensed under CC BY-SA 4.0.

Periodic acid Schiff staining of esophageal candidiasis

periodic-acid-schiff-staining-esophageal-candidiasis-600x400

Figure 2. Periodic Acid Schiff Staining of Esophageal Candidiasis

Periodic acid Schiff (PAS) staining (dark purple) demonstrates fungal morphology in esophageal candidiasis. Esophageal Candidiasis, PAS Stain by KGH is licensed under CC BY-SA 3.0.

  • 1-2-glycols  –  red or dark purple
  • Nuclei  –  blue

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are usually satisfactory, although glutaraldehyde should be avoided.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into periodic acid for 10-30 minutes.
  3. Rinse well with tap water.
  4. Rinse with distilled water.
  5. Place in Schiff’s reagent for 10-30 minutes.
  6. Wash off with distilled water.
  7. Wash well with tap water for about 10 minutes.
  8. Counterstain with Mayer’s hemalum for 2 minutes.
  9. Wash well with tap water until hemalum is blued.
  10. Dehydrate with ethanol, clear with xylene and coverslip using a resinous medium.

Notes

  • Glutaraldehyde fixation leaves free aldehyde groups attached to tissues, which causes an overall positive reaction. These groups may be stopped from reacting with an appropriate procedure such as the aniline-acetic aldehyde block.
  • The tap water wash at step 7 is necessary to develop the red color. Within limits, the longer the wash the darker the color.
  • Originally, it was recommended that the Schiff’s reagent be washed off with dilute sulfurous acid (the sulfite rinses). Since water recolors Schiff’s reagent, it was believed that a water wash could lead to false positive results. It is now known this is not the case, provided the Schiff’s reagent is removed quickly and the sections do not stay in water contaminated with it for extended periods.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
March 30, 2023
Love0

Gomori’s Methenamine Silver for Glycogen and Fungi

By Aldehydes, Carbohydrates, Metal Impregnation, Metal Impregnation, Silver, Protocols, Stain Target, Stain Type
Protocol

Gomori's Methenamine Silver

for Glycogen and Fungi

15
steps
10
materials
print

This method is also known as Grocott’s or Grocott-Gomori’s methenamine silver.

Materials

  • Chromium trioxide, 5% aqu.
  • Neutral red, 1% aqu. or Light green SFy, 0.2% in 0.2% acetic acid, or Progressive hemalum and eosin
  • Sodium bisulfite, 1% aqu.
  • Sodium thiosulfate, 2% aqu.
  • Yellow gold chloride, 0.1% aqu.
  • Stock Methenamine silver
    MaterialAmount
    Methenamine, 3% aqu.100mL
    Silver nitrate, 5% aqu.5mL

    Shake until the precipitate redissolves. Silvering of the container indicates deterioration.

  • Working Methenamine silver
    MaterialAmount
    Stock Methenamine silver25mL
    Distilled water25mL
    Borax*0.1g

    Make just before use and preheat to 50°C.

    *Or 2 mL of a 5% aqueous borax solution (Grocott)

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with 5% chromic acid (chromium trioxide) for 60-90 minutes.
  3. Rinse well with tap water.
  4. Bleach with sodium bisulphite for 1 minute.
  5. Rinse well with tap water.
  6. Rinse with distilled water.
  7. Treat with methenamine silver solution at 50&degC. until impregnated (up to 3 hours)
  8. Wash with distilled water.
  9. Tone with 0.1% gold chloride solution for 5 minutes.
  10. Rinse with distilled water.
  11. Fix in 2% sodium thiosulphate for 5 minutes.
  12. Wash well with running tap water.
  13. Counterstain with light green, neutral red or a light H&E.
  14. Rinse with tap water.
  15. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Oxidisable carbohydrates, including glycogen and fungi  –  black
  • Background  –  as counterstained

Notes

  • Methenamine is also known as hexamethylenetetramine and hexamine.
  • Borax is sodium tetraborate. Grocott’s modification adds the 0.1 g of borax as 2 mL of a 5% aqueous solution.
  • Aqueous solutions of chromium trioxide are usually referred to as chromic acid. Ten minutes in a 10% aqueous solution will usually give the same result as 60 minutes in a 5% solution.
  • Toning is a variable step. Untoned sections give dark brown material on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black material on a pale grey-brown background. Others tone longer (a few minutes) to produce black material on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.
  • This method depends on a similar principle to Bauer’s chromic acid Schiff method, but in which the aldehydes produced by oxidation reduce a silver solution instead of combining with Schiff’s reagent to form a red compound. Consequently, those materials which are red in a Bauer’s stain will be black in Gomori’ stain, i.e. it is not specific for glycogen but will demonstrate any carbohydrates which can be oxidised to aldehydes, including fungi, and itis often used for that purpose.
  • In a similar method Hayashi, Tome and Shimosato recommended that thiosemicarbazide should be applied to the section after oxidation. Thiosemicarbazide has the formula H2NNHCSNH2. The hydrazine group (H2NNH-) combines with any aldehydes present. The thiocarbamyl group (-CSNH2) is a more powerful reducing agent than the aldehydes it replaces and reduces the methenamine silver solution more rapidly and with higher contrast.Immediately following step 5:
    • Place sections in 1% aqueous thiosemicarbazide for 10 minutes.
    • Wash well with tap water, and carry on from step 6.
  • It is well known that metallic azides can be explosive. However, thiosemicarbazide is not a simple metallic azide. The MSDS says:
    • Flash Point: n/a
    • Lower Explosive Limit: n/a
    • Upper Explosive Limit: n/a
    • Unusal Fire and Expl.rds: none identified

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by The Blakiston Co.
    Republished by Robert E. Krieger Publishing Co.
  2. Drury, R A, and Wallington, E A, (1967).
    Carleton’s histological technique., Ed. 5.
    Oxford University Press, London, England.
  3. Hayashi, I., Tome, Y. and Shimosato, Y., (1989)
    Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner.
    Stain Technology, v 64, p 185.
March 30, 2023
Love0

Llewellyn’s PATS (Periodic acid, Thiosemicarbazide, Schmorl)

By Aldehydes, Protocols, Stain Target

Protocol

Llewellyn's PATS

(Periodic acid, Thiosemicarbazide, Schmorl)

10
steps
5
materials
print

This technique demonstrates carbohydrates in a manner analogous to that of the periodic acid Schiff reaction, but gives a blue product.

Materials

  • Periodic acid, 1% aqueous.
  • Thiosemicarbazide, 1% aqueous
  • Schmorl’s solution
    MaterialAmount
    Ferric chloride, 1% aqueous, fresh30mL
    Potassium ferricyanide, 1% aqueous, fresh4mL
    Distilled water6mL

    This solution should be used fresh. Prepare immediately before use.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidize in 1% periodic acid for 10 minutes or longer.
  3. Rinse well with water.
  4. Place into 1% thiosemicarbazide for 5 minutes.
  5. Wash well with running tap water to remove all traces of thiosemicarbazide.
  6. Place into freshly made Schmorl’s solution for 10 minutes.
  7. Wash well with water.
  8. Counterstain with nuclear fast red.
  9. Rinse well with water.
  10. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Oxidizable carbohydrates – blue
  • Reducing substances – blue
  • Nuclei – red

Human kidney stained with periodic acid, thiosemicarbazide, Schmorl's reaction 400x magnification.

Human kidney stained with periodic acid, thiosemicarbazide, Schmorl’s reaction 400x magnification.

Human liver stained with periodic acid, thiosemicarbazide, Schmorl's reaction 400x magnification.
Human liver stained with periodic acid, thiosemicarbazide, Schmorl’s reaction 400x magnification.
Human intestine stained with periodic acid, thiosemicarbazide, Schmorl's reaction 100x magnification.
Human intestine stained with periodic acid, thiosemicarbazide, Schmorl’s reaction 100x magnification.
Human intestine stained with periodic acid, thiosemicarbazide, Schmorl's reaction 400x magnification.
Human intestine stained with periodic acid, thiosemicarbazide, Schmorl’s reaction 400x magnification.

Notes

  • It is well known that metallic azides can be explosive. However, thiosemicarbazide is not a simple metallic azide, it is a carbazide (-C=N-) and is not explosive. It is safe to use.
  • Reducing substances which may be present are also coloured blue. This includes melanin and enterochromaffin. Others may also be seen.
  • If acid hydrolysis is used instead of periodic acid oxidation (as in Feulgen’s nucleal reaction), nuclei are coloured blue.
  • The Schmorl’s solution is from Lillie’s modification of Schmorl’s ferricyanide reduction method for tissue reducing substances.
  • There is a modification of this technique for fungi.
  • Thiosemicarbazide has a hydrazine group at one end of it’s molecule and a thiocarbamyl group at the other. The hydrazine group combines with any aldehydes generated by periodic acid oxidation, and in so doing attaches the thiocarbamyl group to the carbohydrate. The thiocarbamyl group is a more powerful reducing group than aldehydes and rapidly reduces ferricyanide to ferrocyanide, which is immediately trapped by the ferric salt to form prussian blue at the site.
  • Thiosemicarbazide: (hydrazine–thiocarbamyl) = H2NNH–CSNH2

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Hayashi, I., Tome, Y. and Shimosato, Y., 1989
    Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner.
    Stain Technology, v 64, p 185.
  2. Lillie, R.D., (1954)
    Histopathologic technique and practical histochemistry Ed.2
    Blakiston, New York, USA.
  3. Llewellyn, B. D., (2014)
    Thiosemicarbazide-ferricyanide reduction for the histochemical demonstration of aldehydes in tissue sections.
    Biotechnic & Histochemistry, v 89, p 228-31.
March 30, 2023
Love0

McManus’ PAS Reaction for 1-2 Glycols

By Aldehydes, Periodic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type
Protocol

McManus’ PAS Reaction

for 1-2 Glycols

8
steps
5
materials
print

The periodic acid Schiff reaction (PAS) is used to demonstrate the presence of 1-2-glycols, and is consequently an important method in the histochemistry of carbohydrates and the histological demonstration of many structures.

Materials

  • Periodic acid (0.5% aqueous specified).
  • Schiff’s reagent (Coleman’s specified).
  • Harris’ hemalum
  • Light green working solution (0.2% aqueous Light Green diluted 1:5 with distilled water)
  • Ammonia water (water with 3 drops concentrated ammonia per 100 mL)

Tissue Sample

6 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory, although glutaraldehyde should be avoided.

Protocol

  1. Bring sections to distilled water via xylene and ethanol.
  2. Digest using a diastase, hyaluronidase, or sialidase procedure.
  3. Oxidize in Periodic Acid for 5 minutes.
  4. Rinse in distilled water.
  5. Place in Coleman’s or another Schiff’s Reagent for 15 minutes.
  6. Wash in running water for 10 minutes to develop the pink color.
  7. Counterstain with one of the following:–
    1. Harris’ hematoxylin for 6 minutes, then
      1. Wash in running water and transfer to 1% acid ethanol for 3-10 quick dips
      2. Transfer to 1% acid ethanol for 3-10 quick dips
      3. Wash in distilled water
      4. Dip in ammonia water to blue the sections
      5. Wash in running water for ten minutes
    2. Light green working solution for 10 seconds.
  8. Dehydrate with ethanol, clear with xylene, and coverslip using a resinous medium.

Expected Results

  • 1-2-glycols  –  red
  • Nuclei  –  blue
  • Background  –  green (if light green used)

Notes

  • Light Green is better used when delineation of fungi is required.
  • Tap water and ammonia decolorize Light Green, so proceed directly to dehydration.
  • Glutaraldehyde fixed tissues will have a non-specific positive background staining. This must be blocked before step 2.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. McManus, J. F. A., (1946)
    Stain Technology, v23, p99.
March 30, 2023
Love0