[Histonet] PAS with Glycogen Digestion - Solution Not Working

[Mickie Johnson]

Diastase powder will dissolve in any water, but requires some calcium ions
to function. Thus it will not perform in distilled water, but will perform
well using plain tap water. Also, the diastase powder from sigma will work
well if only a very small amount of the powder is used. We always used just
enough to barely cover the bottom of a glass coplin jar and then fill with
tap water. I would expect 0.5 grams in 50 mls of water is probably too much.
Try using one tenth that amount or 0.05 gm in 50 ml. When there is too much
diastase in solution, enzyme conformation is inhibited and it  does not
work. Spokane water is quite hard. It always worked well at room temperature
for 20 minutes.

Mickie
 
Mickie Johnson, B.S., HTL(ASCP)
Mohs Histology Consulting Services, LLC
  & Mohs Lab Staffing
2507 S. Manito Blvd.
Spokane, WA 99203
509-954-7134
FAX   509-624-3926
Web: www.mohshistogyconsulting.com & www.mohslabstaffing.com 
Email: mickie25 <@t> netzero.net
 
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Friday, October 24, 2008 8:38 AM
To: histonet <@t> lists.utsouthwestern.edu; McKnight, Tanisha
Subject: Re: [Histonet] PAS with Glycogen Digestion - Solution Not Working

Tanisha:
Being an enzyme, you cannot use diastase in distilled water. You have to
prepare a diastase buffer at pH7 to dissolve the diastase and do the
digestion at 37ºC during 30 minutes. That will work.
Please, do not try to use saliva!
René J.

--- On Fri, 10/24/08, McKnight, Tanisha <tanisha.mcknight <@t> covance.com>
wrote:

From: McKnight, Tanisha <tanisha.mcknight <@t> covance.com>
Subject: [Histonet] PAS with Glycogen Digestion - Solution Not Working
To: histonet <@t> lists.utsouthwestern.edu
Date: Friday, October 24, 2008, 11:26 AM

Hi All:
 
I am in the process of working up a PAS Staining protocol. The PAS stain
is working fine, but I am unable to get my tissue to digest. I am using
.5g of Diastase in 50ml of DI water. I tried heating the solution in a
water bath for 30 min, 1 hour and 1.5 hours and it still looks the same
as the one without digestion. Could I be using the wrong Diastase? If
so, does anyone have any suggestions for a new product?
 
Thanks,
 
Tanisha Neely, HT(ASCP)
AP-Histology/Specimen Management
 <mailto:tanisha.mcknight <@t> covance.com>  



 


      
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