I have a ear biopsy sample to process for histological analysis. I've processed the tissue according to classic paraffin embedding protocol but I'm not 100% satisfied. If anyone has suggestion or some tricks, it will be very usefull. Thank You
I have problems with sectioning. Once i have good section I'll start staining. I never did this procedure with ear biopsy so I would like to know if there are some special trick I should consider.
It is very difficult to give any advice without knowing the detailed problems. There are many possible difficulties regarding sectioning. Too hard tissue, loose knife or blockholder, insufficient processing, thin-and-thick-sectioning, other sectioning artifacts.
do a google-search for "Artifacts, Faults and Failures: a Review, from Leica", that shows you some points.
I Agree with Miss Lang. You have to see the time of this material stay in formol, the time of process in alcohol, xilol. How many bath did you do in alcohol, xilo and paraffin? How many times? if you want I send you my histological protocol for you test. Best regard.
Thank you for your answer.
I'll look for the Leica review, it will be useful.
Dear Ana, if you can send me your protocol I can compare with mine and try it. thanks.
The protocol I've used first time was quite long (3 bath in each alcohol -30 min, then xilol 30-60 min, 2 h baths in paraffin) ... after this sample was too hard for sectioning... Last time I've tried a different protocol (24 h fix, 30 min each alcohol, xilol-etanol bath, xilol-paraffin bath, then 1 h baths in paraffin) and it seems to work better but section are not perfect.
About sectioning, I used new blade and cut at 5 um using a Leica microtome. After that I did some ee staining to see morphology and I had a quite good morphology of cartilagine layer but the other layer were not so good and compact.
thanks for help
Dear Federica,
Follows my protocol:
Setting time (it's very important) in formaldehyde 4% during 8 h (The size of your material is very important for the formaldehyde penetration).
-Overnight in alcohol 70;
- 2 bath in each alcohol - 40 min (95º, 100º)
- 2 bath in each xilol - 40 min
- 2 bath in each paraffin - 40 (60ºC)
- including new paraffin
*Some points will be need consider: temperature during the addition of the paraffin; the time of use of your baths bateria.
I hope help you.
best regards
Ana Flávia
I use and also proposes sinking in epoxy resin, which is used for electron microscopy. Then cut to a slow-speed diamond saw and further diamond grinding discs.
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