Fixed frozen mouse brain sections crack after thawing, repeatedly - (Apr/06/2013 )

Hi all,

I've got a set of mouse brains that were fixed with PFA through cardiac perfusion. We fixed them in 4% PFA overnight, then 15% sucrose ON, then 30% sucrose ON. Subsequently they were frozen down with powdered dry ice and stored at -80C until cutting.
Cutting was performed on a Leica Cryostat (OT-25C, CT-25C) and mounted on superfrost plus slides with PBS. One brain took about 2 hours and was afterwards air dried at room temperature for half an hour.
Slides were then stored at -80C.

Here's where the problem started:
I took the slides out of the slide box and let them air dry. After a couple of minutes they are all cracked and detach from the slides. I had seperate slides for forebrain and cerebellum. The cerebellum sections are fine, it's just the forebrain which is useless.

I've used that very same protocol many times before on neonatal rat tissue and it worked just fine. I almost never had any significant freezing artifacts or tissue detaching from slides.
It's my second try with that mouse tissue and both times there was the same problem. I thought that there must have been another freeze thaw cycle when someone else transferred my slide boxes. This time I'm quite sure that this didnt happen.

Please help. I'vent found anything on the topic and would very much appreciate advice


thanks,
Damian

I'm sorry I can't help you as I've never used your protocol, maybe you could try something else? We do things a little different. We fix in 4% PFA overnight, then put them straight into 30% sucrose, after 3 days the brains will sink to the bottom which means they are ready to cut. During this time they are stored at 4C. I cut the brains and store the sections in cryoprotective medium at -20C for long term storage. We stain free floating sections and don't mount onto the slides until the staining is over. Our tissue never actually gets frozen so we've never had that problem.

Hi Damian,

Your brain sections cracked maybe a result of not being dehydrated properly in the sucrose solution.

For adult mouse brains, it takes at least 3 days for dehydration to complete - i.e. the brain sinks to the bottom of the tube in 30% sucrose.

For neonatal brains, your sucrose procedure is long enough but definitely not long enough for the adult brains.

Try again soaking the brain till it sinks.

Cheers,

Wah Chin

Hi,

I am having a problem. I have sections of a 10 days muse brain. For staining, I need to use citric acid (20 min, 95C). After taking the slised from the water bath, the tissue is detached and/or folded. This does not happen with sections from an adult mice.

Any idea??

Cheers,

N