I have rat brain samples, that I fixed with 4% PFA but on sectioning they come out very brittle and fall apart. I stopped sectioning because of this. I am looking for a way to recuse the rest of the brains that I haven't sectioned.
Is there a way to remove the excess PFA and still get good sections from this?
yes. good question. vibratome is very specialized tool and requires specialized expertise. I suggest 1. change the humidity inside the cryostat, 2.try warmer temperature (e.g., -15 C rather than -25C), 3. change the blade to have a very sharp blade -examine it under the microscope, it possibly has notches, from previous cutting. Use brand new blade. 4. cut thicker sections, 30 -35 microns, and do floating sections, and mount after IHC. 5. yo can try carefully thawing one brain, and washing for 2 days or longer in PBS, PFA fixative is partially reversible (compared to glut), the tissue clearly was overfixed (unless this is your standard procedure). Very cold temperature inside a cryostat and bad blade can also cause tissue to be ruined. Good luck
Xin Ren Yes These are frozen sections. And I believe I made a bad batch of PFA. Because the sections after those came out good on sectioning.
Igor Nasonkin Thanks for the suggestion. I will give this a try. Is there any published protocol or this is from experience? I am going to try this with one of the samples and report back.
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology
- Sectioning