[Histonet] Wrinkling causes in the staining process?

[Geoff McAuliffe]

Hi Lawrence:

    I agree with John Kiernan's comments and I have several points to 
add. If you are cutting with a glass knife you may have small cracks (as 
John mentioned) because your knife is not as sharp as necessary for 
optimum sections. A second possibility is that the xylene-based mounting 
medium is reacting with the epoxy section and causing wrinkling. Many 
people use a drop of the same epoxy resin the sections are embedded in 
as a mounting medium.

Geoff

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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>


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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff <@t> umdnj.edu
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