check if the antibody works at all on frozen, PFA-fixed sections. Transcription factors usually/frequently require antigen unmasking, which is easy to do on paraffin-embedded sections but hard to do on delicate frozen sections. Autofluorescence may be a result of over-fixing, aldehyde groups on tissue cause this, and one can use 0.1M Glycine to quench autofluorescence, or 0.1% (w/v) sodium borohydride. Lack of specific staining is a different issue. Using enzymatic unmasking (protK or trypsin) may be tried to loosen 3D structure of a transcription factor, but this needs to be done carefully to avoid destroying your sections. Google antigen unmasking methods on frozen PFA-fixed sections. Check if the antibody you bought has been actually used for generating crisp clean data on PFA-fixed froze sections, maybe you need to buy a different antibody. If it was shown to "work well" on histological sections, check if these were paraffin-embedded or PFA-fixed frozen sections. If it works in ICC, check if the sections were fixed by methanol or PFA. Last, doing floating sections will enable better penetration of an antibody. Yet, if you have no signal at all - the critical question is if this antibody is suitable for PFA-fixed frozen IHC. Of course, we have to assume that your antibody has been kept in good condition (in frozen aliquots or in 50% glycerol). Synaptic systems is a reliable company producing many good antibodies for neuroscience research

Agree with Igor O. Nasonkin ,

PFA fixation for 4 hrs will definitely affect your antigen and as you know PFA- fixed Frozen sections are much more fragile than paraffin sections and they do not tolerate harsh antigen retrieval protocols. Why do not you go directly to OCT embedding without prior PFA fixation ::that what I usually do and never have a problem with antigen masking. Or you may shorten your fixation time in PFA 10, 15 , 30 min or maximum 1 hr.

Please refer to this protocol:

https://med.nyu.edu/research/scientific-cores-shared-resources/sites/default/files/frozen-tissue-preparation-with-sucrose.pdf

Hallo Igor, the cfos antibodies from Synaptic Systems are working on PFA fixed tissue. They are also working on mounted non fixed kryosections. After PFA Fixation I recommend to treat the 30µm thick sections in a free floating way. The advangae of this method is that the antibodies can reach the sections from both sides. 0,2% Triton X-100 in PBS as a delution medium helps the antibody to bind even on deeper laying Epitops. And if you use biotinylated secondary antibody together with fluorophore labeled streptavidin then you are working with a high sensitive System which allows you higher dilutions of Primary antibody. We use in our lab cfos from guinea pig and rabbit. I will add some images to give you an Impression of this antibodies an how they look like. Good luck and best reagrads !

Try using the c-Fos antibody from CalbioChem (ref PC38T) I prefer it to Synaptic Systems even though nothing will replace the Santa Cruz one.

Thanks for all your answers.

We could solve our problem by only fixing for an hour in PFA and then keeping the spinal cords for one hour in PBS before transferring to sucrose.

Thanks again!

Constanze