[Histonet] dehydration after staining

[Rene J Buesa]

Gudrun:
  You cannot overdehydrate, you either dehydrate completely or do not dehydrate completely.
  Many (many) years ago the Zeiss microscopes used to have in their Abbé condenser the ability to slide sidewise their field diafragm, and by doing so you were able to obtain "obliquous" illumination = some 3D effect.
  To me the problem is of very different difractive index between the dehydrated tissue and the clearing agent, perhaps enhanced by a=some missalignment of the pathologist's microscope.
  It seems to be an optical artifact. I think that the pathologist shoudl try to use field (Koehler) illumination and determine if the effect continues. I think he is using too much light and a too close diafragm.
  René J.

Gudrun Lang <gu.lang <@t> gmx.at> wrote:
  Hi all,

Is it possible to over-dehydrate the slides after staining?



We do our regular HE-stain with increasing ethanol-concentrations for 1-2
min and then clear in butyl-acetat before coverslipping with Pertex and
glass-coverslips.

One of our pathologists complains about an glassy-3D-appearence of the
tissue, especially of collagenfibers and mamma. The effect is most visible
in his microscope, which is the newest (2002), but only with the 4er and
10er objective. When he switches the condensor in, the effect is gone, but
there is only the small bright space. He compared it to older slides
(cleared in Xylol or Limonen, coverslipped with film or glass) and saw the
same effect in a milder way. The other pathologists don't see the same with
their (older) microskopes.

Could this be a matter of the microscope or a matter of clearing? Or a
matter of wrong handling of the microscope?



I would be happy for any input.



Gudrun Lang







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