Santa Cruz c-fos antibody in brain tissue

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Nimoy
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Santa Cruz c-fos antibody in brain tissue

Has anyone had any problem optimising the Santa Cruz c-fos antibody for immunohistochemistry in brain tissue?

Galicola
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You should be more specific

You should be more specific in your question so we can help you.
What are the problems that you encounter (background, no staining, other)
what controls did you use?
how many different conditions?
etc...

Nimoy
Nimoy's picture
My primary antibody is the

My primary antibody is the polyclonal affinity pure c-fos antibody from Santa Cruz... The secondary antibody I used was a biotinylated donkey anti-rabbit antibody from Jackson Immuno Research... I'm getting virtually no specific staining with very high background at 1:5,000 concentration... I have used both positive and negative controls.... I have increased concentration, blocking with donkey and goat serum... I also remade the buffers, the DAB chromogen... I obtained fresh primary antibody from the company, as well as new ABC kit.... The tissue was originally fixed in 4% paraformaldehyde, cryosectioned into 30um sections.... All that results is a stain with poor signal-to-noise ratio..... Any other suggestions welcome!

Galicola
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Well, sorry, I have no idea

Well, sorry, I have no idea how to help, except using a differnt Ab or a different approach.
However, I found this article and in their methods they say
"We were compelled to quantify only in Layers II/III because the immunostaining pattern with the Fos antisera is most pronounced in these layers. [...] Three frames in each section stained with either our cat Fos antibody or the Fos antibody from Santa Cruz were screened. [...] Because of a very low signal:noise ratio, the computer-aided image analyzing system could detect only background staining and no Fos-positive nuclei."

Tim79
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What part of the brain are

What part of the brain are you staining? Which method of IHC do you use: glass mounted staining or free floating? I've used the same protocol and Abs with free floating and don't have these problems.