Re: Immunohistochemical staining on frozen muscle

From:Neil Hand <mpznhand@unix.ccc.nottingham.ac.uk>

Debra

We use several antibodies supplied from Vector on frozen muscle biopsies.
Our sections are fixed in cold acetone at 4C for 20 mins, thoroughly
air-dried and an LSAB technique applied which include hydrogen peroxide and
serum blocks.  Sections are stained for 1 hr in primary ab and ChemMate
reagents are used as we have a Dako Techmate immunostainer.  Generally
there are no problems with the main ones which include Dys 1 (1:10), Dys 2
(1:5), Dys 3 (1:25), adhalin (1:10), g-sarcoglycan (1:800), emerin (1:400),
merosin (1:6000) dysferlin 1:300) and spectrin (1:100) although when time
permits I want to reassess some.  Certainly dysferlin & g-sarcoglycan are
not as strongly stained as some of the other abs., and some abs are
"cleaner."  We use normal muscle as controls.

Hope this helps.




Neil Hand,

Histopathology Dept,
Queen's Medical Centre,
University Hospital,
Nottingham NG7 2UH,
England, UK




You wrote:
> I'm relatively new to the world of histology.  I'm the Neuromuscular
> technician at the Medical College of GA in Augusta.  My question pertains
> to Immunostaining of muscle biopsies on patients that may have muscular
> dystrophy.  Therefore, I'm only doing dystrophin, sarcoglycan, merosin,
> and occasionally dysferlin.  The problem that I'm experiencing is too
> much background staining and in the case of the gamma-sarcoglycan and
> dysferlin they're not working at all.
> I generally use the dystrophin kit supplied by Vector.
> Since the tissue thats used is small in quanity, I have been doing the
> procedure manually.
> The procedure used is:
> primary antibody (l hour) dilutions 1:300.
> rinse in TBS buffer (pH7.6) 3 x 10 minutes
> secondary antibody ( 1 hour) dilution 1:100.
> rinse in TBS buffer (pH 7.6) 3 x 10 minutes.
> DAB ( 4 minutes)
> counterstain harris hematoxylin.
> This is the protocol that Vector sends with its kit.
> Please let me know of any techniques, etc that will reduce the background
> staining

____________________________________________________________________
Neil Hand
Department Histopathology, University Hospital, Nottingham NG7 2UH.
work : Tel: (0115) 924 9924 extension 43725
____________________________________________________________________





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