On Thu, 26 Aug 1999, NORNAHAR KASSIM wrote:

> Dear Histonetter,
> 
> For those who have done Dystrophin stain(Immunoperoxidase)on muscle tissue.I 
> need your help to solve my problem....
> 
> I use Ab from Novocastra to stain Dystrophin 1,2&3 with dilution
> Dys 1 - 1:5 , Dys 2&3 - 1:10 for 1 hour at RT and secondary Ab I use
> Rabbit Anti-mouse Immunoglobulin with dilution 1:100 for 1/2hr at RT.
> (I use Tris buffer to dilute all Antibodies)
> Then I develop with DAB for about 1-2 minutes,wash,counterstain with
> Haematoxylin ,dehydrate ,clear and mount as usual.
> 
> The problem is fibrous tissue also stains up quite strongly(background 
> staining)make us difficult to interpret/differentiate whether it's a 
> positive staining of Dystrophin or background staining.
> 
> Could anyone of you helps me to solve this problem... please??
> T.Q
> 
> FROM:
> NORNAHAR KASSIM
> MEDICAL LABORATORY TECHNOLOGIST
> DEPT. OF PATHOLOGY
> UNIVERSITY OF MALAYA
> KUALA LUMPUR
> MALAYSIA.
> 
> 
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Nornahar Kassim,
I run Dystrophin on frozen muscle using NovoCastra's Dystrophin 1,2 and 3
also.  Dys 1 at 1:5, Dys 2 and 3 at 1:20.  When I ran them by hand I would
incubate all at 30 minutes RT.  I now use the Dako Autostainer and I
incubate at 15 minutes RT. 
That seems to be the only major difference
between the runs that you and I complete.  Your secondary sounds close to
what I did when I ran them by hand, [Vector's ABC kit (Horse anti-mouse)]
incubated 30 minutes, (their dilution was about 1:100)
Everything else sounds like the rest of my protocol too.
I get consistently good results.  Our neuropathologists think so too.

So maybe you only need to shorten your times.  Good Luck.

Dana Settembre
Immunohistochemistry Lab
Pathology Department
University Hospital
Newark,  New Jersey