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-   -   Bubbles under post-fixed mouse brain sections? (http://www.molecularstation.com/forum/immunohistochemistry-forum/86417-bubbles-under-post-fixed-mouse-brain-sections.html)

sdalin 09-11-2012 02:40 PM

Bubbles under post-fixed mouse brain sections?
 
Hi all, I'm having a problem with my brain sections that I hope someone can help me with!

We have mouse brains that we post-fix in 4% PFA for ~48H, then cryoprotect in 30% sucrose until the brains sink (usually a day or two). Then we embed the brains in OCT and store them at -80C until they are ready for sectioning. I use a Leica cryostat to section them and put the sections on superfrost+ slides.

Before we started postfixing (we used to use fresh frozen sections but were losing the antigens we were interested in) the sections transferred to the slides very nicely. Now though, it seems that the section and the OCT adhere to the slide at different rates resulting in bubbles and wrinkling of the sections. This has a very negative impact on staining as antibodies and other reagents get stuck under the wrinkles, and morphology can become obscured by wrinkles.

We have tried cryoprotecting in 20% sucrose and soaking the brains in OCT at 4C for 30M prior to embedding, however the sections are still wrinkling.

Has anyone seen this problem before? Any suggestions of what else I can try?

Thank you!

haisond 03-11-2013 10:05 PM

Re: Bubbles under post-fixed mouse brain sections?
 
How thick are your slices? 30 minutes in OCT is not enough time to penetrate your tissue. Try soaking them in OCT under vacuum to solve the issue with air bubbles. Wrinkling could be attributed to temperature differential between the microtome blade, your block and the temperature of the cryostat. Allow everything to equilbriate to same temperature setting (place blade, knife, and block in cryostat for at least an hour prior to processing). Alternatively, you can fix the brain by perfusion with 4% PFA, this will clear out all the blood and fixed the brain tissue from the inside out rather than from the outside in as by immersion fixation. Your tissue will be much cleaner with perfusion fixation. There are a lot of good perfusion fixation protocols out there. Also, if you are cutting sections 20 microns or greater, try using a vibratome instead of cryostat. Vibratome makes the sectioning process much easier and with consistent results from slice to slice i.e morphology etc.


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