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.
As Robin said, most likely are due to differences in the perfusion, even if both were transcardially perfused, some perfusions go better than others. Things you can check during perfusion to get an idea of their quality are: (1) has the liver got cleared from blood? (2) has the animal had strong muscular contraction movements? (3) is the brain stiff/hard or is it soft, and is it rather white-ish or pinky-ish?
One thing you could try is to analyze the only the top 1-5% of the histogram in the images you acquire. It might be that using such a threshold you can identify and separate the noise from the signal.
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