[Histonet] why PAS-H staining instead of H-E for testes??

King, Curtis - RAS cking <@t> rallansci.com
Mon Jun 26 08:41:15 CDT 2006

When I did sperm staging I processed Bouins fixed testis to GMA block and
cut 1 micron thick sections. I then stained with PASH and had wonderful
results. My thoughts are that you will not see the acrosomes well enough to
stage them on a 4 to 5 micron thick paraffin section. If I remember
correctly the GLP regulations require GMA processing for sperm staging.

Hope this helps,
Curtis D. King, HT(ASCP)
Tech Rite Consultant
Richard Allan Scientific
4481 Campus Drive
Kalamazoo, MI 49008

Phone: 800-522-7270 ext 672
Fax: 296-372-2734
E-Mail: cking <@t> rallansci.com

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Malcolm
Sent: Monday, June 26, 2006 9:19 AM
To: PKamalavenkatesh <@t> wockhardtin.com; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] why PAS-H staining instead of H-E for testes??

Saw the below email.  In conducting life history studies I have used H-E to
characterize stages of spermatogenesis. IS there an advantage to PAS-H over
H-E?  I assume you must be able to see something you can't with H-E!  Or is
this specific for rats?  I do amphibians and reptiles!  
A New Journal Published in Partnership with Partners in Amphibian and
Reptile Conservaiton
and the World Congress of Herpetology.
Malcolm L. McCallum
Assistant Professor
Department of Biological Sciences
Texas A&M University Texarkana
2600 Robison Rd.
Texarkana, TX 75501
O: 1-903-223-3134
H: 1-903-791-3843
Homepage: https://www.eagle.tamut.edu/faculty/mmccallum/index.html


From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of
PKamalavenkatesh <@t> wockhardtin.com
Sent: Mon 6/26/2006 12:36 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] PAS-H staining-rat seminiferous epithelium-doubts reg

Dear Histonetters,

This  is  with regard to PAS-Hotchkiss staining of rat seminiferous tubules

for  staging  of  spermatogenesis.  This  is  the protocol I have followed.

Testes  got  fixed  in  Bouin's for 24 hrs and washed with 70 % alcohol and

stored  in the same. This protocol I took from histonet archives (Thanks to

Bettina  Hutz,  Research  Assistant,  Department of Discovery Biology Orion

Corporation, ORION PHARMA)


3x xylene                             each min. 3 minutes
2x ethanol 100%                               short
ethanol 90%                           short
ethanol 80%                           short
ethanol 70%                           short

Periodic acid  (oxidation)            5 minutes
70% ethanol                           1 minute
Reduction solution                            1 minute
70% ethanol                           1 minute
Schiff�s reagent                            20 minutes
Running tap water                             10 minutes
Mayer�s hemalaun                    3 minutes
Running tap water                             10 minutes

Dehydration & mounting
ethanol 70%                           short
ethanol 90%                           short
2x ethanol 100%                               short
3x xylene                             short
Mount with xylene based medium

 But  I  am  feeling that the staining quality is inferior (staining of the
acrosomes)  to  correctly  detect  the  various stages. I have few pictures
published  by  the  European  Society  of Toxicopathology, which are simply
fantastic.  I  don't know where I am failing. Now my question is any of our
members  are  having  a  protocol,  which  they  feel working very well and
anybody  can  able  to  send  me  the  photos  of various stages of the rat
spermatogenesis  they  got. This will enable me to compare my staining with
that of others.

Pre clinical safety assessment division
New Drug Discovery- Biology
Wockhardt Research Center
Please Visit our New Corporate Web Site www.wockhardt.com
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