Immunohistochemical staining on frozen muscle

From:Debra Lloyd <>

Hi everyone,
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 

Debra L. Lloyd, BS
Neuromuscular Pathology Lab
Medical College of GA  
phone: (706) 721-2312

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