We isolated mice brain after perfusion with chilled PBS, followed by fixation in 4%PFA (24hr) and 30% sucrose dehydration until sank to bottom of tube. Later, brain samples were cryo preserved . While sectioning in cryotome at 30um. We observed that in some brain samples, sections are very porous or web like. Is this due to preservation issue or can it be due to treatment conditions.
can i still use these sections for IHC/IF?
Hi Krishna Singh Bisht
* The sucrose perfused brain tissues should be immediately frozen in liquid nitrogen. The porosity may arise if its slow freezed.
* The porous structure in the tissue may arise due to temperature changes in the cryotome. Keep the temperature at -25 degrees and keep the blocks in the cryotome half an hour at least prior to sectioning. Hope it works.
Thanks
Samir
2 factors are important.
One is as Samir mentioned the time of deepfreezing the tissue blocks. Liquid Nitrogen is fine as well as dry ice or drop the temperature of blockholder up to -45°C. The duration of cryoprotection in sucrose is also important. I recommand 3-5 days for mouse brauns and upto 7 days for rat brains.
With a Nissl's stain (cresyl violet) you can check the quality of your sections .
Hello,
I usually post-fix for 24hrs and put in sucrose for 24hrs after which mouse brains sink.
I had absolutely no problem in conserving fixed brains in 20-30% sucrose at 4°C for 1-2 years and IF staining still worked perfectly. Once cut, I keep my sections either free-floating in 24 well plates in cryopreservation buffer at -20°C or on microscope slides at -20°C or -80°C.
This porosity could come from bad perfusion or post-fixing. Your PFA should be fresh (no more than 1 month old) if not, fixation won't be as good.
Thank you for your suggestions @ Samir Ranjan Panda , Ute Neubacher Louis-Charles Béland .
Dear Krishna,
I agree with Louise-Charles that the problem may be bad perfusion or post-fixation. I looked at your photo of the sections, and in some ways it looks like it may be that the blood vessels are larger in diameter than normal, and this seems to be the case in all the sections on that slide. If that is true, it could be that the perfusion pressure was too high, which will artificially enlarge the blood vessels.
I am also not sure I understand your fixation procedure. If you are perfusing with PBS, do you not also perfuse with fixative afterwards? For brain tissue, this is much better than immersion fixation for good tissue morphology.
Another possibility is that you need stepwise sucrose cryoprotection. I am usually able to able to place my adult mouse brains into 30% sucrose after a good fixation. But after certain fixation protocols I have had to use the 10%-20%-30% stepwise treatment, making sure that the tissue sinks to the bottom of the tube at each step. I also post-fix and cryoprotect at 4 C.
Good Luck!
Jill
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