[Histonet] Encountering the same problem with wrinkled rat brain
nfournier <@t> sasktel.net
Mon Aug 21 15:06:59 CDT 2006
I apologize first for the long email; however, I wanted to include as much information as possible.
We are still having a problem on what I have posted previously on regarding wrinkling of rat brain sections after
mounting. Our tissue was fixed with 4% paraformaldehyde for 48 hrs prior to being sectioned at 50 micron on
a Vibratome 3000. The vibratome bath contains 0.1 M PB at 2 to 4 degrees C. Typically, we have processed
the tissue for various immunohistochemistry free-floating protocols, such as BrdU, doublecortin, NeuN,
GAD67, etc. We noticed that after the staining was complete we would encountered severe wrinkling when the
tissue was brought through alcohols and stained with cresyl violet. We have altered a variety of parameters
such as as our cresyl violet protocol, using gelcohol in mounting solution, using Fisher Superfrost/Plus versus
gelatin subbed slides, etc. However, we found that the wrinkling was minimized, if after mounting the sections,
they were throroughly dried (by using a Kim Wipe to wipe up residual 0.1 M PB. We use 0.1 M PB, pH=7.4 for
mountng) and allowed to sit for at least 2-3 hrs (vertical position) before being placed in a small oven set to 38
to 40 degrees C for overnight. (I have also left tissue in the oven for at least 2 to 3 days before running the
slides through alcohols, xylene and coverslipping with no observed differences in staining quality). None of
the other changes were associated with any real differences in tissue wrinkling. We have also used non-
immunohistochemistry processed tissue and have encountered the s ame wrinkling problems.
I don't think my method for mounting is incorrect, but just to let you know, I typically fill a petri dish with 0.1 M
PB and using a paint brush I gently float the sections on to a coverslip after which I lightly transfer the section to
a dry slide (e.g., Fisher Superfrost/Plus) and gently absorb residual PB with a Kim Wipe.
Now I thought the issue was the result of the sections not being dry enough prior to coverslipping; however, I
have started to second guess this. A colleague of mine, who has far more experience than I do using vibrating
microtomes, encountered the same problem. They were using 40-50 micron thick fixed rat brain sections
through a free-floating procedure for immunofluorescence. After the staining was complete, the sections were
mounted on to Fisher Superfrost/Plus slides and antifade medium was applied. Significant wrinkling was still
observed. They have also tried mounting sections, drying them, and leaving them in the fridge overnight prior
to coverslipping. Wrinkling was still observed. My colleague does not want to dry placing the slides in the
drying oven because he feels this might effect the fluorescence. Finally, they have used a different vibrating
microtome and the issues were not observed.
We do not experience these problems with cryosection tissue and are now starting to wonder if the problem
may be the result of using a vibrating microtome. Has anyone encountered similar problems using these types
of sections and determined methods to fix this?
Any help is appreciated,
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