[Histonet] PAS with diastase

[Tony Henwood]

Try the method described in 

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): 153-154.

We have used it for over 5 years and have had no problems.

Method as follows:

Document Procedure:	
Diastase Removal of Glycogen


Pathnet Code:	S DPAS

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 (Fluka 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.


Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318



-----Original Message-----
From: MICHELE M [mailto:immunogal3 <@t> yahoo.com] 
Sent: Thursday, 19 August 2004 7:17 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] PAS with diastase


Hi All,
 
We're looking for some help with our PAS with diastase stain.  For some
reason, we can not get it to work. The PAS works fine but the diastase does
not seem to be working.  We are using diastase of malt from Fisher and a
0.1M phosphate buffer at pH 6.0 from Polyscientific.  Our tech also tried
using distilled water with the diastase instead of the buffer. The diastase
solution is heated in a waterbath at 37 degrees for one hour. We have used
liver, colon and esophagus tissue controls.  If anyone has any ideas what
might be the problem, please let us know.
 
Thanks,
 
Michele

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