I dissect the animal, intracardially perfuse with 0.9% Saline, then with few ml of 4% PFA intracardially. Then i remove the brain and fix it in 10 % formalin for 2-3 days after which i embed the brain in paraffin and process it. During paraffin sectioning, i consistently observed there is vaccum in hippocampal area, which is like a shrunken hippocampus. I could literally see a cavity while sectioning in a microtome. Although i can get hippocampal sections, little beneath the actual area.. Can anyone suggest any reason for this shrunken/Vaccum, which i encounter in hippocampal region? Does this cavity arise due to the Circle of Willus, which directly supply hippocampus, which is situated just above the basal midbrain????
HI,
Immediate thing which comes to my mind is the pH of the saline. Pls check the pH.
We generally perfuse animals transcardially with 4% paraformaldehyde in 0.1 M phosphate buffered saline, pH 7.2, and the tissues were post-fixed after with 4% PFA again.
You can post-fix using 4%PFA instead of formalin and check it.. Hope it helps.
It is a perfusion issue, which needs to be improved/fixed. can be fixed. Good luck!
Dear Pandi-Perumal,
Can you please help me which step am i going wrong?? Or How can i fix the perfusion issue ??? Please help
Over the years, I have worked in the wet labs. However, I have moved to clinical field. You may try the method proposed by Santosh. A decade ago, I was interested in looking at some specific brain cells with a particular staining. I requested some brain specimen from overseas. The PI was a busy person. He requested one of his junior associate (as the expert has gone on leave) to perfuse and ship the brain specimen. The fact that the perfusion was not done correctly, we had problem fixing, transferring, and sectioning. When we looked the mounted slides, the sections were too bad. The tissues come off easily in every procedure. We end up discarding the entire shipment due to bad quality of perfusion. I thought I could tell you that there are issues such as this one, which you need to overcome!
As I am not an expert on perfusion, you could wait for others to give their inputs. Good luck!!!
if you are fixing using PFA by perfusion, why dont you perform cyropreservation and cryosectioning
Hi Ray!!!
I think that could be because of not proper fixation, tissue might have putrefied. the problem may be with perfusion or may be PFA or preparation of PFA.
I see two major possible reasons of your problem:
1) As mentioned by Santosh, check your PFA solution pH. I normally dissolve PFA in standart phosphate buffered saline and the pH is around 7.1-7.2.
2) Try another stock of dry PFA - yours may be not pure enough or just contaminated.
Also try to fix the braain in the same 4% PFA solution instead of 10% formalin.
Good luck!
Hello,
This is a tissue fixation and/or tissue processing issue. The brain tissue was perfused and placed in either pH balanced 4% paraformaldehyde or 10% neutral buffered formalin(NBF) for fixation. As fixation is critical for tissue preservation, allow the fixative to penetrate at a rate of 1mm per hour. The fixation of your animal brains for 2-3 days in 10% formalin might be sufficient. However, different animals require longer periods in a fixative. You should visualize the tissue for consistency and uniformity of fixation prior to proceeding to the next step.
The brain must go through a tissue processor prior to tissue embedding in paraffin. The tissue processor will complete cycles of dehydration with an increasing concentration of alcohols, clearing with xylene or substitute, and infiltration with paraffin. According to your steps above, you went from tissue fixation directly to tissue embedding. Then during microtomy you noticed the sunken regions of the hippocampus. If so, the brain tissue did not complete the tissue processing step.
The sunken region of the hippocampus may not only be a fixation problem but also tissue processing problem. You can not go directly from fixation to embedding. The hydration of the fixative will not allow the paraffin to properly infiltrate the tissue. Your problem can be prevented by following the steps to create tissue slides from paraffin blocks.
Fixation-tissue processing-embedding-microtomy-staining-coverslipping
Hope this helps and Good Luck,
Brian Johnson
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