Has anybody out there stained for ECM (adsorbed if poss) w/o secondary nonspecific binding

From:Gail Donegan

Hello All,

I am immunostaining for adsorbed extracellular matrix proteins (laid down 
by fibroblasts and smooth muscle cells) and have had persistent 
non-specific binding of not one but three secondary antibodies

- one using the alkaline phosphatase/ABC kit and,
- two using rhodamine conjugated secondary antibodies. I've tried blocking 
serums from multiple species, multiple dilutions and various incubation 

I may have not read up on the subject sufficiently, but the journals I have 
read which say they have stained for, say, fibronectin or collagen, often 
have used intracellular primary antibodies. This has begun to suggest to me 
that they stained intracellularly for collagen etc because they suffered 
the same fate as me when staining extracellularly.

If I'm jumping to the wrong conclusions, I'd be delighted to know.

Has anybody out there stained for extracellular matrix proteins using 
extracellular antibodies. Better still, has anybody out there stained for 
adsorbed or deposited extracellular matrix?

By the way....A Big Thank You  for the many tips and recommendations I've 
had from all at Histonet regarding non-specific 2o antibody binding - 
despite many people being away at the congress at this time.

Yours hopefully,

Gail Donegan

Gail Donegan
Department of Clinical Engineering,
The University of Liverpool,
Duncan Building,
Daulby Street,
L69 3GA.

Tel:    +44(0)151 706 5198
Fax:    +44(0)151 706 5803

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