Author Topic: Help with mouse brain cryostat slices (fall off from superfrost plus!)  (Read 2566 times)

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Offline AArwen

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Hello!

I'm recetly starting a project that involves a lot of staining of mouse brain, however I'm not familiar with cryostat..basically, I'm learning it and the worse thing is that in my lab no one has a strong background on it. So I'm here to kindly ask you some suggestions. :)

I perfuse mice with PF 4%, then I post fix in PF 4% O/N. I wash three times in PBS 1X and then I post-fix in scurose 30% for a day and a half (I wait until brains sink at the bottom of a 50 mL tube). I them embed them in OCT and freeze them in isopentane cooled in liquid nitrogen. Once frozed I put them in -80C.

I cut brains (10 microns, usually) in a cryostat. I usually set the temperature at -22C/-25C. I've been told to add a drop of PBS1X on the superfrost plus glass before picking up the brain slice because it is supposed to help the tissue to adhere (is it true?...I'm not that convinced, to be honest). Once I finish cutting I let the slices air dry O/N (or at least for 7 hours) at RT. Then I put them in -80C. At this point slices look very fine, even my boss and other people much more familiar with cryostat sectioning told me that they do look good - one was even really impressed when I told her it was the second time ever that I was cutting. However when I start the staining process (I just put the slices in PBS1X in a coplin jar with nothing else) they tend to fall of from the glass. Some of them just detach a little, other just fall off completely. I really do not understand why this happens.  :( Staining turns out pretty nice, however morphology isn't that great due to the "falling of"

Do you have any suggestions?

Thanks so much,
Arwen