Re: [Histonet] Wrinkling causes in the staining process?

From:John Kiernan

Dear Lawrence D Lanberg,

You don't specify the type of tissue or plastic,
or the sizes of the sections. Are these tiny
little squares cut from blocks also used for
ultrathin sections for EM? Or are they bigger
sections, intended only for light microscopy? Has
the tissue passed through glutaraldehyde, osmium
tetroxide etc (for EM) or some simpler fixation
for LM? You mention ribbons, a term usually
applied to adhering paraffin sections coming off a
steel knife edge. In your case, does ribbons mean
lines of sections floating on the trough behind a
glass or diamond knife?

If these are semi-thin sections of specimens
prepared for EM (embedded in a cross-linked epoxy
resin), the lines that you describe might be
cracks rather than wrinkles. Air drying is usually
OK for semi-thin epoxy sections, but cracking
happens when sections of any thickness air-dry too
quickly, whatever the embedding medium (if any).
In your procedure, air drying after 100% alcohol
(Step 5) could well be the culprit, and it's not
needed. Move the slide from the last 100% alcohol
through 2 Coplin jars of xylene, and apply the
coverslip, using Permount or any other resinous

If this doesn't solve the problem, look at the
sections with a microscope (wet and dry) at every
Find out when the wrinkles or cracks develop.
Optical trickery can reveal physical defects that
are invisible with the ideal (Kohler) illumination
that we use for transparent, stained slides. 

To trace a physical artifact, change first the
illumination. Lower the condenser a bit. Close
down the substage diaphragm. Intrude a 5-10mm
object (a pea on a pin) between the field iris and
the condenser, a bit off-centre. That trick can
provide an almost Nomarski effect.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
Lawrence D Lanberg/O/VCU wrote:
> I am hoping that a microtomist far more experienced than I might know a
> likely cause for wrinkling in semi-thin (990nm)plastic sectioning, after
> the cutting process. The wrinkles will be profuse on one section, but its
> neighbor on the slide may be completely perfect. All sections will come
> from the very same ribbon. The wrinkles appear as extremely thin, spidery
> lines in an otherwise beautful section.
> Through much investigation into the matter, we are reaching the conclusion
> that the cause must be in the staining or mounting process and not the
> cutting, floating or drying process.
> 1. I dry the slides overnight on a low-to-very low heat. *The sections
> appear very nice after this step, under microscopic examination.
> 2. Next day I cover these with Toluidine Blue and set the stain, into the
> sections, briefly on a moderate heat. 70 - 80 degrees Celsius.
> 3. I rinse lightly with deionized water and then a gentle wash of 70% EtOH.
> 4. Complete destaining by submerging in Coplins of 100% EtOH.
> 5. Air dry.
> 6. A drop or two of xylene on the stained sections, then place face down
> onto a cover slip that has a BB-sized drop of Permount.
> Is anything about this process likely to cause a wrinkling? If so, is there
> an alternative step that's worth trying? Any input will be greatly
> appreciated. Thanks so very much.
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