I saw a poster at a conference that presented data suggesting that by altering the pH and/or the volume of 4% PFA during transcardial perfusion it is possible to tailor the fix for optimal labeling with individual antibodies. Does anyone have any experience with this?
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, 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...
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.
We tried several conditions of fixation changing PFA concentration (0.1% to 4%), pH (7.5 to 9.5), postfixation time (0h to 4h), fixative (PFA, Glutaraldehide, Alcohol...). I can tell that most of them have an impact in the final results, some Ab will work in almost all conditions others need fine tunning. Our standard protocol is flush with Saline followed by PFA 4% pH9.5 in Borax buffer, 3h postfix in PFA, ON in sucrose-PBS 15%.
As a starting point you can take a look at Dako Handbook Fixative chapter.
http://www.ihcworld.com/_books/Dako_Handbook.pdf
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