PAS Staining

This protocol is modified from http://www.ihcworld.com/_protocols/special_stains/pas_diastase_ellis.htm

Alpha-amylase, also known as diastase, is an enzyme commonly present in saliva. The action is to cleave the a-glucosidic 1,4 linkages of starch or glycogen to yield water soluble sugars (maltose) and dextrins. These water soluble sugars are then washed from the section.

Periodic acid (HIO4) solutions oxidise 1,2-glycol groups 2(H-C-OH), producing a dialdehyde, 2(R-CHO). By treating with a pale straw coloured solution of leuco fuchsin (Schiff's reagent), the resultant dialdehyde is demonstrated by the colour precipitation of an insoluble magenta coloured complex (aldehyde fuchsin).

Technical Points

1. Not critical, however fixation by aldehyde addition, eg glutaraldehyde, will produce non-specific general PAS positivity. 2. (step 2) - glycogen is removed from the visualisation by pre-treating the section with amylase. 3. (step 6) - Schiff's reagent deteriorates rapidly if not kept in a closed container. When a pinkish discolouration appears, discard the reagent. 4. (step 7) - Washing not only removes any excess reagent from the section, but also promotes the development of the rich magenta colour. Too gentle washing will result in a strong artefactual red stained background due to the action of the powerful dye basic fuchsin, formed from the destabilisation of the leuco fuchsin by loss of sulphurous acid to the watery environment. 5. (step 11) - Over differentiation can lead to the eventual decolourisation of PAS positive material. Method

1. Bring sections to distilled water. Note: Omit steps 2 and 3 for PAS stain only 2. Treat sections with amylase solution 20 mins 3. Wash well in running tap water 4. Treat with periodic acid .5 mins 5. Wash well in distilled water 6. Stain with Schiff's reagent 10 mins 7. Wash in fast running tap water 3-5 mins 8. Stain nuclei with haematoxylin 1 min 9. Rinse in running tap water 10. Differentiate with acid alcohol 11. Wash in running tap water 12. Blue nuclei in Scott’s 13. Rinse in running tap water 14. Dehydrate, clear and mount.

Results

                       PAS positive materials....................magenta
                       nuclei........................................blue
                       erythrocytes................................pale pink

Glycogen is removed by diastase/amylase and is therefore unstained in a PASD

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Reagent Formulae

1. Diastase solution 0.5%

     a-amylase         0.25g
     distilled water   50.0ml

Dissolve the enzyme in the water with gentle shaking. Prepare fresh. a-amylase preferred is Sigma a-amylase type VI-B, product number A3176. The Sigma preparation is stated to be extracted from porcine pancreas.

2. Periodic acid 0.5%

           Periodic acid      2.5g
           Distilled water    500 ml

Store refrigerated

3. Schiff's reagent

a)  basic fuchsin dye (CI 42500)        6.0 g
     distilled water                            1200.0 ml
     N hydrochloric acid                     60.0 ml
     sodium metabisulphite               12.0 g

Dissolve 6 g basic uchsin in 600ml of distilled water. Bring to the boil and allow to boil for a few minutes. Cool to 50°C and add 60 ml N HCl. Cool further to 25°C and add 12g sodium metabisulphite (Analar). Allow the solution to stand and bleach for 24 hours in the dark. & After 24 hours add 5 to 6 g of activated decolourising charcoal and shake for approximately one minute.

Filter rapidly through coarse filter paper. The solution should be clear. If it possesses colour then repeat the charcoal step./font>

Finally add 600 ml of distilled water. Store in a dark bottle in the fridge.

b) Pre-prepared Schiff’s reagent - Australian Biostain Pty Ltd,PO Box 1407,Traralgon, VIC.

• Using PAS Staining System: 395B-1KT

1. Deparaffinze and hydrate sections to deionized water.
2. Immerse slides in Periodic Acid Solution for 5 minutes at room temperature (18-26C).
3. Rinse slide in several changes of distilled water.
4. Immerse slides in Schiff's Reagent for 15 minutes at room temperature (18-26C).
5. Wash slides in running tap water for 5 minutes.
6. Counterstain slides in Hematoxylin Solution, Gill No. 3, for 90 seconds.
7. Rinse slides in running tap water.
8. Dehydrate, clear, and mount sections in toluene or xylene based mounting media.

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/2/395.pdf