Which method is better to prepare brain frozen sections?
A) Directly, after sacrifice freeze as rapidly as possible at -80ºC without fixative. Fix before immunohistochemistry / IFI.
B) Fix in 4% PFA por 24 h and then, sucrose and finally preserve at -80ºC.
Hello Joana,
if you do not need unfixed tissue for special staining methods then you should fixate it. The best way is to perfuse the animals with 4% PFA, post fixation in 4%PFA for 24 -48 h, cryoprotection in 30% sucrose. You can store the brains in sucrose without deep freezing at -80°C. Keep them in the fridge at 4°C until you start sectioning. If you like to use free floating sections for immunohistochimestry you can put a slections of section in cryo-tubes containing a solution made of glycerol and 30%sucrose (equal parts). You can store this sctions at -20°C for months and/or years. Sections which have mounted directly on slides store in slide boxes at -20°C.
Hi Joana Antonela Asensio
* During sacrifice perform cardiac perfusion in animals with PBS and 4% PFA. That would help out in clearing the brain of any excess blood or clots.
* Extract the brain from mice and then store in 4% PFA for 48hrs at 4 degree.
* Following fixation, keep the brain tissues in gradient sucrose (10, 20 and 30%) in each solution until the tissues settle down. (to prevent bacterial contamination 0.1% sodium azide can be added)
* Now the sections can be cut and stored in -20 or -80 for long term use.
Thanks,
Hello,
in any case I recommend to freeze the brains (mouse or rat or small human brain pieces) in liquid isopentane at -80° C. So it will keep his right form.
Some persons put the specimen in alu-foil and then put it in the freezer. In this case the brains get a flat form! Not good!!!
How can we 100 % neutral phosphate buffer solution which is also used as preservative for brain
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