[Histonet] Wrinkling causes in the staining process?
|From:||Lawrence D Lanberg/O/VCU |
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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