I do free floating sections for IHC and i would like to eliminate xylene in the process. Is it possible to leave out the clearing in xylene before coverslipping? Or will it have an affect on the transparency etc.?
Liisa Jacobsen
Dear Lisa,
It will be nice if you could kindly make your question a bit more clear. In fact, I have miserably failed in understanding your question.
With best wishes,
ARB
Dear Ashis
I will try to make my question more clear.
I mount free floating tissue on slides and let lave them to dry. When they are completely dry I let the slides soak in xylene for some minutes before coverslipping with mounting media(I use DPX). I would like to know if I can leave out the step with xylene or will it have an affect on how the staining of the tissue will look in the microscope (bright field)?
If your tissue was well fixed, you can mount your sections from water to the slide (not buffer because of the possibility of salt crystals etc). Make sure it is flat with no wrinkles and then let it dry out. You can then coverslip with a xylene-based media. Do one or two to see if the quality is what you would expect. Sometimes going through the Xylene will also affect the intensity of the stain, so this is another variable to consider. Good luck
Dear Liisa,
I would be careful with removing xylene, I have experience with tissue drying out much much faster under the coverslip when xylene is excluded and getting dry tissue artefacts. If you mount from water then remember to use a waterbased mounting medium. I would as a minimum do some test stains as Frank suggests.
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