Periodic Acid Schiff / Diastase
Classification: carbohydrate stain
Mechanism of staining: histochemical
Purpose: group I carbohydrates (neutral polysaccharides— glycogen, simple polysaccharides, non-ionic homoglycans)
Positive controls: liver, kidney, muscle
Periodic Acid Schiff Positive
Liver
Positive - substance not lost during fixation, oxidation product not soluble, tissue should yield aldehyde upon oxidation, and sufficient aldehyde should be present for detectable end product.
Periodic Acid Schiff Positive
Kidney
end product magenta
Nuclei - blue
REAGENT |
PURPOSE |
MECHANISM OF STAINING |
SOURCE OF ERROR |
Diastase |
Negative control |
Glycogen hydrolyzes with addition of amylase
from saliva or commercial product from malt. Negative control tissue is
treated with diastase to confirm glycogen. |
Omitted: No negative control. Positive components will be
demonstrated. |
Too short: Some carbohydrate positive components will be
demonstrated. |
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Too long: No effect. |
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Periodic acid |
Oxidation |
Ionic bonding due to oxidizer (periodic acid), which opens up
aldehyde groups and further reacts with Schiff reagent to form color. |
Omitted: No aldehyde groups will be produced, therefore no
reaction occurs and no carbohydrates will be demonstrated. |
Too short: Weak or no reaction occurs and no carbohydrates
will be demonstrated. |
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Too long: Background staining. |
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Too strong: Tissue non reactive because
carboxylated groups formed. |
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Schiff reagent |
Leuco dye o Basic fuchsin o Sulphurous acid o HCl o Thionyl chloride |
Histochemical -binds to aldehyde groups with chromophores to
form a colored end product. |
Omitted: No reaction with target compound. |
Too short: Target compound not stained properly. |
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Too long: No effect |
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Old Schiff reagent: pink deposits Yellow Schiff reagent: acridine added. |
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Harris hematoxylin or Alcian blue |
Counterstain – to demonstrate nuclei |
Ionic bonding |
Omitted: No nuclei demonstrated. |
Too short: Pale reaction. |
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Too long: Nuclei obscured, background staining. |
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Acid alcohol |
Differentiator |
|
Omitted: Background staining. |
Too short: Background staining. Under differentiation. |
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Too long: Over-decolorization, no nuclei details seen. Over
differentiation. |
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Scott’s tap water |
Bluing agent |
|
Omitted: Nuclei will be red and no details seen. |
Too short: Nuclei will remain red. |
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Too long: No effect. |
Periodic Acid Schiff Positive
Liver
Periodic Acid Schiff Positive
Kidney - basement membrane stains magenta.
PAS Positive
Thyroid follicles demonstrate magenta staining thyroid colloid.
Periodic Acid Schiff Diastase
Liver
Negative control tissue treated with diastase to confirm glycogen.
Kupffer cells are resistant to diastase and will appear magenta in colour
Periodic Acid Schiff Diastase
Liver shows bile pigments as light brown.
Periodic Acid Schiff Diastase Negative
Liver with resistant fungi appearing purple.
Special Considerations
Troubleshoot only counterstain, a histochemical reaction is permanent.
Negative control tissue to confirm glycogen and is always used.
Any formalin or mercury pigments should be removed before oxidation takes place.
References
Officer B. HIML 251 Lecture notes: Carbohydrate stains: Periodic Acid Schiff and Perls Prussian Blue, January 14, 2009.