[Histonet] Amylase Digestion for glycogen

Tony Henwood AnthonyH <@t> chw.edu.au
Mon Feb 7 00:40:01 CST 2011


Our amylase digestion procedure is quite simple (see: Mangan, V-M, Farago, V., Kelly, M., Henwood, A.F., (2002) "An Amylase Reagent with a Long Shelf Life for the removal of Glycogen from Tissue Sections" J. Histotechnol 25:153.) See below for the method:

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.


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.

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Ruppert, Amysue
Sent: Saturday, 5 February 2011 11:03 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Amylase Digestion for glycogen

 We are looking to switch from malt diastase digestion for glycogen to Amylase digestion. I have the new protocol worked up, but one of the Pathologists I work with would like to have an idea of how many labs out there are using Amylase instead of malt diastase for their PAS/D method.
 If you use amylase for the PAS/D method, could you please let me know who you are and the institiution?
 Much appreciated.

amysue ruppert
Histology lab
Marshfield Labs
Marshfield WI

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