Maybe the abs with the background are too strong and maybe the abs with no
staining are too weak. Have you used normal muscle as controls for testing
the titres of these abs? You should include the Spectrin ab in your panel as
this checks the integrity of the muscle fibre membranes, this should always
be positive.  The procedure you wrote is an indirect method, we use a
3-stage method (DAKO LSAB2 kit). Also the buffer rinses seem rather
excessive, we do x2 buffer rinses for 1 min each. You could always contact
Vector, as they supplied the kit, for help.

Bob Quilty
Dept of Neuropathology
Frenchay Hospital
Bristol   UK

----- Original Message -----
From: Debra Lloyd <dlloyd@neuro.mcg.edu>
To: <histonet@pathology.swmed.edu>
Sent: Wednesday, May 02, 2001 12:35 PM
Subject: Immunohistochemical staining on frozen muscle


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