Xylen is only necessary for deparaffination of paraffin embeded samples. For H&E of cryoslides you should fix your slides also before staining, then rinse in destilled water and go on.

ok, thank you.

I completely agree with Gudrun. Staining sections with H&E needs fixation in either neutral formalin or Bouin's fluid and doesn't need crystallizing. However, if the latter was necessarily performed, then you will need to fix the slides and rinse in distilled water before staining.  

Gudrun answered your question, but  to be even more precise - here is the sequence: frozen section > fixed > washed > stained for H&E > washed > alcohols  > xylene or xylene substutute > coverslip.

You may avoid xylene by using an aqueous mountant, but if you do IHC on consecutive sections you probably process at the end via xylene and permanent mountant - so just do the same.

It seems that you are at the beginning of your study so two additional points:

Staining for H&E is simple, but timing depends on fixative and choice of reagents for the staining (different haematoxylins and eosins). If you search online e.g.on "IHC world" website you will find a good and detailed description of many histochemistry and IHC methods.

If your post-fix your tissue after freezing - the cytological details are not as good as when you fix tissue before freezing. If you alredy have material for your study unfixed/ Y frozen then immediate fixing of  sections when cutting is essential for preservation of your tissue for IHC. Post-fixing gives you an advantage of choosing your fixatives and not being committed to only one. Depending on what antigens you wish to disclose in your samples you may fix consecutive frozen sections in different fixatives, e.g. some antigens need antigen retrieval when tissue is fixed in formalin, but  do not need antigen retrieval when fixed in alcohol and acid based Carnoy's fixative or another non-aldehyde fixative.

Good luck with your work.

Thank you so much for the well detailed answer . Actually I am not in the beginning of my study but it is true to say that I did not do H&E with frozen samples. I only did IF (Immuno fluorescence). The Objective of this experiment is to check the morphology of the spleen, and the Granuloma tissue (lymphofollicular and non follicular structure), so after labeling the slides I will scan them with programme NDP (nano zoomer digital pathology). Again thanks

Sorry, that's my pathology background showing - we start every research on tissue with 'orientational' H&E staining, followed by a panel of immunos, scanning and image analysis - hence my mistake. Again - good luck with finalising your study.

All the responses have described well the procedures to follow and the pitfalls to avoid. I want just to insist, as said by Maria,  on the fact that your IHC labeling will be better and more useful when you fix your tissues just after their removal.

Yes Fatiha,

your comment is right: it is either fast fixation or perfusion.

Cheers

This discussion is getting quite long, and veering away from H&E staining, but there is one more point to add about fixation (and cryo-protection) before freezing. One can store safely fixed and frozen samples in OCT for cutting at a later stage, and store tissue and cut  cryo-sections from fixed frozen tissue for much longer at -80degC. Storing unfixed material for cutting and post-fixing is fine only for a fairly rapid work.