[Histonet] Paraformaldehyde fixation questions

[Lewis, Patrick]

After having a lot of problems  using 100% Acetone as a fixative, I am going to try 4% paraformaldehyde in DPBS pH 7.4  for fixation of slides cut from fresh tissues snap frozen in OCT.

My plan is to cut the slides at 5 or 6 uM and dry them for 2 hours in the hood, then fix them in the 4% p-f for 10 minutes.

Then immediately immerse them in Room temp DPBS.

Is 10 minutes fixation long enough?  Would 30 minutes work  better?

Is there any advantage, or disadvantage of using 4C versus Room temp for Paraformaldehyde?


I am concerned about the drying of the tissues, and tissue loss from the slide.

I'm not sure which will be best, drying after cutting or drying after fixing.

Years ago, I vaguely remember being told that you should never dry the tissue after fixing it and then re-wet it.

So I am leaning towards drying after cutting, but keeping the slides wet after fixing.

The goal is for me to get good fixation, not destroy epitopes, and avoid the tissue damage that I get when I fix with Acetone.

Thanks

Patrick.

PS: My brief internet search stated that Fixation occurs faster at higher temperatures (which I knew) , and that Some antibodies are capricious so for them 4C is better. (= IHC myth that corresponds to find what works and then don't change it)

Is it right to assume that 4C is going to be gentler on the tissues than Room temp.


CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.