I am doing immunohistochemistry on auditory nerve fibers in the mouse brain. I am perfusing with 0.1% glutaraldehyde, 4% paraformaldehyde to fix the tissue, with the plan of taking confocal images of the fluorescence followed by EM on the same sections. My issue is very high levels of non-specific fluorescence after treating with the secondary fluorescent antibody, and I can't see the structures we expect in the tissue under the confocal. I have heard that this could result from non-specific binding of the antibodies because of free aldehydes, and I have tried treating the tissue with NaBH4 prior to antibody labeling, but cannot seem to decrease the non-specific fluorescence.
Has anyone dealt with this issue or have a protocol for how to reduce it?
Have you seen these structure before and the problem is new?
Either way, I recommend using the highest quality grade fixatives you can get, buy in ampoules and make the fixatives fresh, filter them through a 20nm pore and store for a week max. (I would filter all of the buffers that come in contact with the sample also)
Its expensive, but when I've had background issues in single molecule microscopy this has helped.
Hey!
If the non-specific background is due to unbound aldehydes, then introducing a step before the labelling, in which you incubate the samples in e.g. glycine solution or some other amino acid might work. The free aldehydes should then react with the amine groups and not be available anymore for the antibodies.
Glutaraldehyde is known to cause trouble in IHC/IF. It can mask epitopes and also causes strong autofluorescence in the tissue. Do you do an antigen retrieval step before staining? That might help if the antigens are indeed masked by the glutaraldehyde.
Hope this helps!
Cheers,
Sven