[Histonet] Encountering the same problem with wrinkled rat brain sections

[N Fournier]

Hi Colleagues,

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,

Thanks,

Neil