[Histonet] PAS-diastase staining

Sun May 31 20:20:00 CDT 2015

We have been making our own diastase solution for years now. Works extremely well. The diastase is very easy to make in house. Here is the protocol for making your own diastase

Principle:	
The enzyme solution is applied to one of two sections of the tissue (preferably consecutive sections) and then both are stained by the PAS method.  The presence and relative amount of glycogen in the sections can be determined by examining the extent of loss of staining in the enzyme treated section as compared with the untreated section.

This amylase reagent has a long shelf life and uses an incubation time of 10 minutes at room temperature. It is suitable for formalin and Brazil’s fixed paraffin sections as well as air-dried and ethanol fixed frozen sections.

Controls:		Liver containing glycogen

Reagents: 		
1.	Amylase Reagent
Warning: Harmful, contains azide – see MSDS
Alpha Amylase from Bacillus Subtilis (Sigma Cat No 10070,)	1g
Oxoid PBS Tablets (Cat No BR14a)				1 tablet
Distilled water							100ml
Sodium Azide							0.1g
This solution, once prepared is stored at 4oC when not in use. A recycled antibody dropper bottle (often used in commercial immunoperoxidase kits) is useful for storage and application.

2.	PAS Reagents (see PAS Stain)
Warning: Suspected Carcinogen – see MSDS

Procedure:

1.	Dewax and hydrate paraffin sections, hydrate frozen sections.
2.	For amylase digestion, place slides on a rack, cover sections with amylase solution and allow to incubate for 10 minutes at room temperature.
3.	Wash slides well in water.
4.	Place slides in 1% periodic acid 10 minutes.
5.	Wash slides well in water.
6.	Rinse slides in distilled water.
7.	Place in Schiff’s reagent 10 minutes.
8.	Rinse slides in distilled water and then wash slides in tap water 3 minutes.
9.	Counterstain slides with haematoxylin, differentiate and blue.
10.	Dehydrate, clear and mount.

Results: 
•	Glycogen is extracted and so loss of PAS positive staining will occur in the enzyme treated section.

•	Mucopolysaccharides are not extracted and so staining will be the same in both sections.

Reference:	 
V-M. Mangan, V. Farago, M. Kelly, and A. F. Henwood (2002) " An Amylase Reagent with a Long Shelf Life for the Removal of Glycogen from Tissue Sections" J Histotechnol. 25(3): 153-4.

Linda Prasad | Senior Scientist | Histopathology
t: (02) 9845 3306 | f: (02) 9845 3318 | e: linda.prasad at health.nsw.gov.au | w: www.schn.health.nsw.gov.au
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-----Original Message-----
From: Roy, Lisa [mailto:Royl1 at LabCorp.com] 
Sent: Saturday, 30 May 2015 1:37 AM
To: Rene J Buesa; Julio Benavides; histonet at lists.utsouthwestern.edu
Subject: Re: [Histonet] PAS-diastase staining

I have been using the spit method for years with great results.  Depar slide to water, add saliva directly to slide for 10 minutes, rinse and stain PAS as usual.  Also American MasterTech has diastase malt that works well.
Lisa

-----Original Message-----
From: Rene J Buesa [mailto:rjbuesa at yahoo.com] 
Sent: Friday, May 29, 2015 11:17 AM
To: Julio Benavides; histonet at lists.utsouthwestern.edu
Subject: Re: [Histonet] PAS-diastase staining

Sigma porcine diastase is very good.A cheaper option is very disgusting but had been done for decades, just spit on the section but that will carry bacteria and, although I have seen doing it, I have never done it.René J.  

     On Friday, May 29, 2015 10:01 AM, Julio Benavides <j.benavides at eae.csic.es> wrote:

 Hi there,

I am trying to do a PAA-diastase in formalin-fixed embedded samples (ovine liver). I would be most grateful if someone could give me a hand in:

-Protocol (enzyme concentration, time of digestion...) for diastase digestion (I am assuming that PAS afterwards is the normal protocol for PAS) -A commercial source for diastase working in these samples. Is the porcine amylase from sigma any good? cheaper options?

As always,

thank you very much for all your comments

Cheers

Julio

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     On Friday, May 29, 2015 10:01 AM, Julio Benavides <j.benavides at eae.csic.es> wrote:

 Hi there,

I am trying to do a PAA-diastase in formalin-fixed embedded samples (ovine liver). I would be most grateful if someone could give me a hand in:

-Protocol (enzyme concentration, time of digestion...) for diastase digestion (I am assuming that PAS afterwards is the normal protocol for PAS) -A commercial source for diastase working in these samples. Is the porcine amylase from sigma any good? cheaper options?

As always,

thank you very much for all your comments

Cheers

Julio

_______________________________________________
Histonet mailing list
Histonet at lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

     On Friday, May 29, 2015 10:01 AM, Julio Benavides <j.benavides at eae.csic.es> wrote:

 Hi there,

I am trying to do a PAA-diastase in formalin-fixed embedded samples (ovine liver). I would be most grateful if someone could give me a hand in:

-Protocol (enzyme concentration, time of digestion...) for diastase digestion (I am assuming that PAS afterwards is the normal protocol for PAS) -A commercial source for diastase working in these samples. Is the porcine amylase from sigma any good? cheaper options?

As always,

thank you very much for all your comments

Cheers

Julio

_______________________________________________
Histonet mailing list
Histonet at lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

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