I want to do rat brain immunohistochemistry, and I find that rats were perfused transcardially with PFA under deep anesthesia in all published papers. But now some people in pathology told me that rats can be decapitated and immediaterly to get brain, after saline washing, the brain fixed to 4% PFA is OK. Should I to do perfusion at first?
Dear Yanli,
Transcardial transfusion has at least two main benefits: (1) fast removal of the red blood cells from the vessels embedded in the brain (2) fast fixation of the tissue to preserve structural integrity. With decapition you will not maximize such benefits. For details of fixation and immunohistochemistry please see my PhD thesis listed below, and also a recent methodological considerations inspired from previous RG questions.
Best wishes,
Refik Kanjhan
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According to my work in brain immunohistochemistry, I prefer either decapitation or deep anaesthesia then transfer the brain tissues as soon as you can to the fixator NBF. As you know immuno-markers are so sensitive against the use of alot of chemicals (e.g. gluteraldehyde in trans-cardiac fixation).
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In our lab we obtained similar immunohistochemical results on rat brain slices of perfused brain or brains that were fixed in 4% PFA immediately after isolation - after the isolation, the brains are cut coronally in half (globus palidus/thalamus region) for faster penetration of PFA, postfixed by immersion in 20% sucrose in 4% PFA at 4°C (until they sink) and stored in 20% sucrose in sodium phosphate buffer at 4°C (add sodium azide for longer storage) until sectioning in cryostat or using freezing-stage microtome (free-floating brain slices). For immunohistochemistry, we use hydrogen peroxide to quench endogenous peroxidase.
Some antibodies won't work after several days in PFA 4%. You should wash and put PBS after 48 hrs and cut as soon as possible.
Dear Yanli,
Picking the best method of fixation is always a trade off between antigenicity and morphological preservation.
I also prefer perfusion fixation with 4% paraformaldehyde to immersion fixation. Paraformaldehyde can penetrate into tissue fairly quickly, but it is not instantaneous. Leica shows this problem with formaldehye and liver, here:
http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/
If you are trying to fix an entire brain, this can be very important because you don't want you cells of interest at deeper levels of the brain to undergo any sort of degenerative changes before the fixative reaches them.
If you think paraformaldehyde might mask your antigen, decapitation followed by quickly freezing the brain tissue (I use powdered dry ice) is useful for producing cryostat sections that can be fixed by any method that is compatible with your antibody. For example, I have used certain antibodies that were only compatible with acetone or methanol fixation, so I had to take this route.
Good Luck!
Jill
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