[Histonet] PAS-H staining-rat seminiferous epithelium-doubts reg

[PKamalavenkatesh <@t> wockhardtin.com]

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)


Deparaffination

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

Staining
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.

REGARDS
Dr.P.Kamalavenkatesh
Pre clinical safety assessment division
New Drug Discovery- Biology
Wockhardt Research Center
India
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