Hi,
For some reason I get much higher c-fos background signal in the rat brain for one animal than for the other (see attached photo). The staining protocol is the same for both specimens, as well as the blocking step (1 hour @ 25°C with 10% NDS). I have repeated the staining protocol several times and the bakground difference is the same time and time again. It is also consistent no matter what part of the brain or brain stem I look at. How to approach this difference? Is this definitely non-specific staining? What do you suggest?
Thanks,
Jan
I am pretty sure that the difference is due to tissue quality. Was the animal perfused? If yes, the animal with the higher background had a suboptimal perfusion, if no, try to switch to intracardial perfusion.
Hi Jan, I don't know how to fix your problem, but is it possible that it's due to tissue collection? For example, were both samples equally fresh before they were fixated?
To see whether it isn't a specificity problem due to bad quality of tissue, have you tried negative controls with only the secondary antibody and/or no antibody at all (naked tissue autofluorescence?)?
I am pretty sure that the difference is due to tissue quality. Was the animal perfused? If yes, the animal with the higher background had a suboptimal perfusion, if no, try to switch to intracardial perfusion.
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