RE: [Histonet] Encountering the same problem with wrinkled rat brainsections
Those of us associated with Vibratome company were concerned with your
problem and have consulted an expert.
"The first thing I would suspect is the coating on their glass slides.
Superfrost isn't good enough for adherence for free-floating sections at
~30 microns. Thaw-mounting from a cryostat onto Superfrost does work
however, presumably because it is in the absence of immersion in buffer.
But Niel also tried gelatin coating. The recipes and batches of this
method are variable and oftentimes unreliable. I would suggest checking
the recipe and protocol, using another recipe or simply using the
poly-L-lysine technique: 1% poly-L-lysine (in water) smeared over a VERY
CLEAN glass slide and allowed to dry for 5 minutes. Baking the tissue
is unnecessary and probably a bad idea for ligand-binding as well as
tissue adherence. Tissue is best allowed to dry naturally - without too
much blotting or using fans, hair dryers, ovens, etc. Overnight at room
temperature is fine but usually it really only needs 1-3 hours. Another
possibility is that Neil's alcohols are in the wrong sequence or
contaminated with water. They should be fresh, and tissue should be
immersed for 3-5 minutes in this sequence: water, 50% EtOH, 75% EtOH,
90% EtOH, 100% EtOH, 100% EtOH again, and Xylene twice. The only way the
vibratome is the culprit that I can see is if it is cutting too thick -
way to thick - like 100 microns too thick. Pretty unlikely without
My money is on the slide coating - and save the oven for Thanksgiving!"
Miles Cunningham, McLean Hospital
Charles W. Scouten, Ph.D.
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300 x 342
FAX 314 522 0377
[mailto:email@example.com] On Behalf Of N
Sent: Monday, August 21, 2006 3:07 PM
Subject: [Histonet] Encountering the same problem with wrinkled rat
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
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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