[Histonet] Mouse staining for ATP

Nails, Felton flnails <@t> texaschildrens.org
Wed Feb 9 09:24:36 CST 2011

 I am looking for a reliable method for staining mouse muscle with ATP'ase pH 4.3, 4.6 and 9.4.
Any help will be greatly appreciated.

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tom McNemar
Sent: Wednesday, February 09, 2011 9:05 AM
To: 'Erin Sarricks'; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] PAS with Diastase Digestion

Spit  for 5 minutes....  old school....

Tom McNemar, HT(ASCP)
Histology Co-ordinator
Licking Memorial Health Systems
(740) 348-4163
(740) 348-4166
tmcnemar <@t> lmhealth.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Erin Sarricks
Sent: Wednesday, February 09, 2011 7:38 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] PAS with Diastase Digestion

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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