First off I would suggest for consistency sake that you not use separate methods of fixing tissue in an experiment. However for some preliminary data or your to develop an expectation of whether your experimentation might work, you can try a couple of things to salvage your tissue. If you're at the antigen is relatively stable you can try to post fix the tissue in the fresh formalin and section as you normally would. Otherwise, you can treat the tissue I crash tissue and flash freeze it. You can then cryo- section it onto a slide once sectioned, you can then post fix and stain in the tissue. When you have blood left in the tissue it is imperative that you use fresh formalin since formic acid 10 react with the blood produced setting a dark and auto-fluorescent precipitate.

Good luck

I don't think, that the treatment with HCl will remove erythrocytes. The blocking of endogenous peroxidase is important to repress the DAB-staining of erys. Then it shouldn't matter in regard to your IHC-protocol.

I am really no expert in animal-preparation, but I would be more concerned about delayed fixation or tissue-damage, if the blood was pressed into the skull/brain. ?

I would prefer phosphate buffered 4% formaldehyde for fixation. With buffered NBF formalin-pigment (heme-artefact) is not expected, and it is recommended for IHC.

If you put the brain or skull in formalin quickly after euthanasia and perfusion, it will be fine, but if it's still in the skull you will need to leave it there for more than 24 hrs for fixation to complete. There is no absolute need for perfusion in brain examination, immersion fixation works fine for general purposes. BrdU IHC can be easily done with immersion fixation, it is not that delicate an epitope.The fact that there is still blood cells within vessels should not impede your technique in any way. Also, I'm not sure why you have an HCl step in the IHC technique, to my knowledge it is not a common step.

Thank you all for your responses, they are all very helpful. To clarify, I immediately extracted the brain and placed it in a 4% paraformaldehyde and 20% sucrose post-fix which I plan to leave for 24 hours, at which point I typically move it to a 30% sucrose solution until it sinks. GUDRUN: For blocking of endogenous peroxidase I typically do a 10 min incubation in 0.5% H2O2. Because this brain is bloodier than I am used to getting, would you suggest a longer and/or higher concentration (of H2O2) incubation (of course, as JUSTIN pointed out, I will then do the same across all animals to keep consistency)? JEAN-MARTIN: are you suggesting that a little bit of blood in the tissue is not as disastrous as I am thinking it is? I have no experience with this, but my advisor tells me that bloody tissue is essentially useless and it should be thrown away (but he is not a histologist). And the HCl step is common to all BrdU protocols I have received from colleagues of my advisor. It is my understanding that the HCl is needed to denature the nucleus so that the anti-BrdU can get to the DNA.

Hi Francis,

 The fact that there are blood cells remaining in the vessels does not make a tissue useless, otherwise pathology would not exist as a discipline. It all depends on the applications of course, and if you have other animals in the same study that are getting perfused, the overall morphology of this one animal might be a bit different. But if the main thing you are doing is looking at cell proliferation, that shouldn't be affected that much by fixation variability. Are your targets of interest in the neuropil, or are they the vessels themselves ? 

As for HCl, I now understand that it's a BrdU-specific thing, so I have no problem with it.

Peroxidase-block: If your protocol has worked with your former experiments, I wouldn't change it. Here we do a 3% H2O2 in water or methanol for 5 min.

You can prove it. Take one section for testing. Go directly after blocking and rinsing into chromogen-substrate-solution. If the ery are all DAB-brown, blocking was insufficient.

Also, even if you have taken the brain out of the skull, I would leave it in formaldehyde longer than 24hours, to allow proper penetration of the fixative. I'm not sure what having 30% sucrose included with the formaldehyde will do to the fixation.