Dear all,
I am going to do some transcardial perfusion using 4% PFA and do a post fixation of the brain O/N.
My question is which the best way to preserve the brain afterwards so we can use it for future experiments?
I intend to slice the brain with the aid of a vibratome, and our aim is to do IHC, EM and also do some WB.
Thank you for your help and I look forward for the replies :)
Kind regards,
Daniel
Hi Daniel,
I would keep them in the fridge, in PBS with 0.05% NaN3 (sodium azide).
FYI, this will be fine for IHC/IF, but for EM you normally would also have a low percent of glutaraldehyde (around 1%, together with the PFA) during perfusion and post-fix. Obviously your vibratome slices will require further OsO4 processing, resin embedding and resectioning but most EM labs can help out with this (you definitely don't want to work with osmium tetroxide if you don't have to, and ultramicrotomy is an art form in itself!)...
And for most WB, you really want to avoid fixation altogether and instead freeze your tissue as fast as possible before moving on to your isolation/lysing/other prep with protease/phosphatase inhibitors added before or during thawing.
Hope this helps!
Best,
Alberto
Hi Alberto,
Thank you for your reply!
I understand that snap freezing the tissue is better for WB, however we have to compromise since we have a limited amount of subjects. I know it is not ideally, but since PFA can be de-crosslinked that is why I am thinking of not using glutaraldehyde (since this one is not possible to de-crosslink, correct me if I am wrong).
If I don't use glutaraldehyde is still possible to achieve good results when doing the EM?
Cheers,
Daniel
Hi Daniel,
The best way would be to embed in OCT and store at -80. For IHC such storage preserves tissue architechture and protein expression. But if you want to do EM then i would suggest that you find another embedding media compatible with EM. The point being that the best way to store tissue post fixation is by embedding to preserve protein expression and storing it at -80 degrees. Do note that OCT frozen brains have to be cut in a cryotome and not a vibratome. Hope this helps!
Arghya
Dear Daniel:
In my experience, the best manner to preserve a fixed brain is perfoming a quick froze in isopentane-liquid nitrogen, and then save it immediately at -80°C. That have work to us, for immunofluorescence and RNA-FISH experiments.
Regarding to the use of this tissue for westernblot, I have never heard about a de-crosslinking, as you sayed. If you need to measure protein levels, just use IHC (or immunofluorescence); these techniques can give you more information about protein levels and location.
Good luck!!!