Dear all,
I am planning to do histology on frozen sections of hard tissues (mouse bones). I treated the samples as following: 4% PFA for 4h, then sucrose gradients (10%, 20%, 30% with 24h each), embed in SCEM, and section it with Kawamoto´s film. Now I would like to try H&E, and Acian blue - nuclear fast red stainings on my frozen sections, but I am not experienced with frozen sections before, so I would be very happy if you could share me a protocol or some tips. Also, I wonder if it is better to place the sections directly on the slide (and do you use coated or non-coated slide?), or transfer them on Kawamoto´s film prior to placing on the glass slide.
Thank you very much in advance, and I am looking forward to learning from you.
Cheers,
Mai
Hi Mohamed Mahdy ,
Thank you very much for kindly sharing the protocols. I´ve tried the protocol for Acian blue - nuclear fast red as above. The staining is fine, but I faced another problem: as I used Kawamoto film for sectioning of undecalcified tissue, final clearing step with xylol made the glue from the film melted and messy.
Would you have any suggestions for this issue?
Hi Mohamed Mahdy ,
Thanks again for sharing your experience.
I applied the protocols for paraffin sections on my frozen sections. They worked nicely, and I excluded clearing in xylene in my final step to avoid the film get dissolved and messy. Also, it´s important to pay attention to temperature during the first washing step as it would help to minimize thawing of the tissue and hence fractures within the tissue. Another point is to gently wash or apply any liquid on the tissue.
I´m quite glad that these points help a lot, and my problems are somehow solved. But if you have other tips working with such type of tissue/method, I would greatly appreciate that :)
Cheers,
Mai
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