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

 

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.

Too long: No effect.

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.

Too long: Background staining.

Too strong: Tissue non reactive because carboxylated groups formed.

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.

Too long: No effect

Old Schiff reagent: pink deposits

Yellow Schiff reagent: acridine added.

Harris hematoxylin or Alcian blue

Counterstain – to demonstrate nuclei

Ionic bonding

Omitted: No nuclei demonstrated.

Too short: Pale reaction.

Too long: Nuclei obscured, background staining.

Acid alcohol

Differentiator

 

Omitted: Background staining.

Too short: Background staining. Under differentiation.

Too long: Over-decolorization, no nuclei details seen. Over differentiation.

Scott’s tap water

Bluing agent

 

Omitted: Nuclei will be red and no details seen.

Too short: Nuclei will remain red.

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.