Hi Justin, The correct way is as you comment. First you have to perform an intracardiac perfusion with PBS solution to wash the vascular system, after that, you have to perform a cardiac perfusion of 4% PFA solution. The volume of perfusion will depend of the weight of the rat. For a rat of 250-300 gr the volume of each solution will be approximately 150- 200 ml.

Hi Justin, in my experience the best way is to: 1. perfuse with 4% PFA, 2. postfix with 4% PFA for 24h, 3. wash 3x 5 min with phosphate buffer or PBS (if no phosphate buffer); 4. transfer to 20% sucrose (with a droplet of sodium azide)

Cutting: keep very low cryostat  temperature -25C or lower, beware of tissue wrinkling; cut at 12um (thinner sections tend to break)

As far as I know the pH of PFA is always 7.4 (physiologically normal). Blood washout should be with 0.9% saline rather that PBS because the phosphates can have an adverse effect on the intended experiment.

The volume of PFA (4% at pH 7.4) following a clear saline run out can only be adjusted to a minimal volume as there is no maximum perfusion...you can't overfix with the same concentration. For mouse, with a clear run out, a minimum volume of 250mls should ensure all those peripheral blood vessels are reached

Thanks for the information.  What I am looking for is information from someone who has experience with advanced methods of fixation. Specifically, I would like to know if by altering the pH or volume of PFA used during fixation, has anyone been able to improve labeling of a particular epitope that may be sensitive to the extent of fixation.  There are reports of fixation with PFA at a pH of 9.4 which was supposed to increase the rate of cross-linking.  I assume that decreasing the pH would have the opposite effect as would decreasing the volume of PFA used.  It seems that with a lighter fix, IHC that normally requires antigen retrieval could be performed without this additional step.  On the other hand, maybe IHC that "doesn't work" with a particular antibody could be rescued with a deeper fix.

Have you tried no fixation? Brain can be tricky if unfixed because it is very soft but it is possible...

PFA is more like cross-linking of amine and if you wish to try a different fixation, how about acetone or methanol to precipitate protein instead of cross-linking? pH trouble might be solved after.

We are interested in preserving the integrity of the tissue so I believe we must use some degree of cross-linking fixation unless we were to frost-mount the sections; however, when we frost-mount, the morphology of the tissue inevitably changes so we prefer fixed free-floating sections for most IHC.