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Author Topic: Fixed frozen section mounting and preserving questions  (Read 4158 times)

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Offline JWG

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Fixed frozen section mounting and preserving questions
« on: September 27, 2012, 11:19:26 PM »
Hi all, I am working with 4% PFA-fixed (perfusion fixed), cryosection mice brain.
We usually perfuse the mice with 10ml PBS followed by 20ml 4% PFA, then remove the brain and soak overnight in 4%PFA, followed by 10%, 20% and 30% sucrose (until sink) before freezing in OCT (in 2-methylbutane cooled on dry ice). We have try thick sections for free floating fluorescent staining (40 um) and it turned out to have autofluorescence problem, so we want to switch to IHC using thinner sections (6-10 um).

The problem we've encountered is that when sectioning at 6-10um thick, we found it pretty difficult to get a nice and flat section if we thaw mount it directly onto the glass slides (they seem pretty dry to attach onto the slides). So we have been putting the section onto a 0.1M phosphate buffer bath and float mount them onto the slides, let them air dry for a couple hours to overnight, then place them into a zip-bag and store at -80C freezer. But, I'm not sure if by floating the section on PB it would hydrate them, and damage the tissue upon freezing at -80C?

Another question, we have been just storing thick section (40um for free floating) in PBS at 4C, and it can only last for up to 2 weeks. Is there a better way to preserve the thick section for free-floating?

Fixed frozen section mounting and preserving questions
« on: September 27, 2012, 11:19:26 PM »