[Histonet] PAS/D digestion problem

[Tony Henwood]

The following is easy to prepare, easy to use and works every time
(shelf life at least 4 months (we run out of it by then):

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.

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.	continue with PAS reaction.

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.

Regards

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




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Josh
Wray
Sent: Monday, 14 April 2008 4:35 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] PAS/D digestion problem


 Hello,


 Does anyone out there have a problem with their digesting of a PAS/D? I
was wondering if I could get some feedback on some peoples methods of
digestion that works well for them to pass along to our Histo Dept.


         Thanks,
          Josh Wray HT(ASCP)
          Esoterics Laboratory
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