Lack of immunostaining at high antibody concentration

From:Jane Radford

Dear Histonetters,

I have a researcher working in our lab that is obtaining unusual 
results with his immunostaining.
The researcher is using a rabbit anti human TRAIL (H257) antibody on 
human melanoma (intransit dermal metastasis) tissue. He has just 
competed a dilution series of the primary antibody (20, 10 and 5 
ug/mL) and found that the highest concentration (20ug/mL) actually 
resulted in no staining at all.  The other two dilutions gave a 
moderate level (10ug/mL) and very faint level (5ug/mL)  of staining 
which is what I would have expected. I would have logically expected 
the 20ug/mL concentration to have resulted in more intense staining.

He made serial dilutions of the primary antibody so we know it must 
be in the solution and other than forgetting to apply the primary 
antibody or other components to the slide in question (which he 
assured me he didn't!) could there be a reason such as inhibition of 
binding of the primary antibody due to it being present in too high a 
concentration, or some other explanation?
Thank you in advance for your help,

Jane Radford

Jane Radford
Histopathology Laboratory Manager
Department of Pathology
University of Sydney
NSW Australia
Ph: 61 2 9351 6152
Fax: 61 2 9351 3429

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