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

[Geoff McAuliffe]

Here are my suggestions for your PAS staining.

1. Make periodic acid fresh each day.
2. What is the "reduction solution" you are using after the periodic 
acid? Why oxidize and then reduce?? Delete this step.
3. Follow the Schiff's reagent with 3 changes of fresh 0.5% sodium 
metabisulfite, 1-2 minutes each. NOT running water. This will make the 
stain more precise.
4. Your dehydration and clearing steps may be too short to remove all 
water and alcohol, there is no reason for one to hurry these steps.
5. Be sure your Schiff's reagent is good, test by putting one drop into 
formaldehyde, it should turn magenta/pink within a few seconds. If not 
make fresh.

Geoff

PKamalavenkatesh <@t> wockhardtin.com wrote:

>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
>Please Visit our New Corporate Web Site www.wockhardt.com
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff <@t> umdnj.edu
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