Nowadays I have experiment to analyze the skin tissue. so we have tried to make paraffin block. tissue size is about 18 x 14 x 9 mm. but we failed to section the paraffin block, just section the paraffin not included skin tissue. why happen to this?
our protocol for H&E staining. it was used PFA 4% overnight, alcohol 30, 50, 70, 90, 100 %, which was distributed with about 30~45 minutes. and then histo- clear instead of xylene during 2 hours and next two phase the paraffin during 1days.
pleases give me advice
Dear Soonjoo, I hope that your fixation was adequate - I use overnight fixation in 4%PFA for skin punch biopsies, for larger samples 24 hours - and your skin sample is thick (I assume that your depth of the block is 14 mm and tickness of skin is 9 mm. In my experiments I use each alcohol (25, 50, 75, 95% for at least 45 minutes and 100 3-times. I used xylene and now Histoclear 3 x 45 min to 1 hour (depending on the size of the sample - for research samples I always check that after clearing in xylene or Histoclear the sample is translucent - if not, the longer clearing solves the problem. Rushing through alcohols may leave tissue not entirely dehydrated and there is a part of the tissue which is not cleared. the only solution then is to revert to the last alcohol and after that - back to xylene. If you were using a processor - I would suggest that you process your sample in reverse order, back to alcohol and repeat final alcohol 3x clearing on the shaker and check that your tissue is translucent before transferring it into wax. The best results for permeating the tissue with wax is if you use oven with vacuum and do not exceed 3 x 1 hour. I have to 'rescue' students' samples from time to time and reversing the process works - but the result is not always optimal comparing to one time processing (the tissue can become harder, a bit more shrunken than the samples processed only once and it may feel more brittle when cutting). Good luck with rescuing your sample.
I did not understand the exact problem however this might help you troubleshooting:
http://www.leica-microsystems.com/fileadmin/academy/2011/95.9890_Rev_A_Difficult_Blocks.pdf
Dear Mustafa, thank you for including this link to Leica paper covering a wide variety of issues with tissue processing. Leica has also a text by the same author "101 Steps to Better Histology" http://www2.leicabiosystems.com/101-steps-better-histology-web which covers dealing with tissue from receiving the fresh specimen to troubleshooting histo-ans immunostaining. You need to register on the website to be able to download it. It is a very good read for students in histology lab, before starting practical part of the project.
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