[Histonet] PAS with Diastase Digestion

[Rene J Buesa]

You CAN NOT use water to dissolve the diastase. You have to use diastase buffer pH7 otherwise it will not work (as in your case).
René J.

--- On Wed, 2/9/11, Erin Sarricks <esarricks <@t> gmail.com> wrote:


From: Erin Sarricks <esarricks <@t> gmail.com>
Subject: [Histonet] PAS with Diastase Digestion
To: histonet <@t> lists.utsouthwestern.edu
Date: Wednesday, February 9, 2011, 7:38 AM


Hi Histonet-



I recently ran a PAS/D stain and had some issues with it.  Both "with" and
"without" slides came out looking the same so I'm guessing my digestion step
didn't work!  I used a Malt Diastase solution (0.5g to 500mL water) for my
digestion.  This is the procedure I used:



1.    Deparaffinize and hydrate to water.

2.    Place the sections labeled “with” in diastase solution preheated to
37˚C for 1 hour.  Hold the sections labeled “without” in distilled water.

3.    Wash in running water for 5 minutes

4.    Place all section (with and without) in 0.5% periodic acid solution
for 5 minutes

5.    Wash in 3 changes of distilled water

6.    Place in Schiff reagent for 15 minutes

7.    Wash in lukewarm tap water for 5 minutes (immediately sections turn
dark pink color).

8.    Counterstain in Mayer’s Hematoxylin for 3 minutes.

9.    Wash in tap water for 10 minutes

10.   Dehydrate starting with 95% ETOH, clear, and coverslip.



I am wondering if my solution possibly got too warm in the oven and hindered
the enzyme activity, or is it possible I left it in too long?  Any tips
would be much appreciated!  Oh, and I have about 300 slides to stain, so
spitting on them is my last last last resort!  Haha!  Thanks in advance for
all your help!







Erin Sarricks, HT (ASCP)

Histology Laboratory Technician

USAMRICD Comparative Pathology Branch

Office: Bldg E-3081 Room 178

E-mail: erin.p.sarricks <@t> us.army.mil
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