I am wondering if anyone can help me. I have some cadaver skin tissues that were processed, however I did not get proper infiltration. I used an extended human process (etoh 70 - 2 hr, etoh 80 - 2 hr, etoh 95 - 2 hr, etoh 100 - 4 hr, xylene A - 1 hr, xylene B - 1 hr, xylene C - 2 hrs, wax A - 1 hr, wax B - 1 hr, wax C - 2 hrs). I am wondering if there are anything protocols for processing that will help with infiltration. Or if anyone has any tips to share for dealing with difficult cadaver specimens (specifically skin).
Thank you all!
Dear Tanya,
How long was the fixation and in what? What is the size of the specimen (WxHxD in mm)? Did you trim before? In majority of the cases your processing protocol should be fine...
The fixation was 4% PFA for 24 hours (give or take). I did not trim the samples. They were all approximately 10x10x5. I've never had this kind of issue with processing cadaver samples before, but my morphology was horrendous.
I think your fixation is too short. Do 48h but not more. You have to trim the sample into slices 3x10x5 mm. Then volume of fixative 10-15x the volume of the sample. Should work normally. Do you do the processing manually?
No, it's an autoprocessor. Also I think the fixation time was an estimate as it came from our gross anatomy lab. So really, who knows. I will definitely try the trimming though.
Thank you so much for your help, Ivan!
Also during infiltration set the infiltration to vacuum, if possible. Brings better results.
If you see a "wet" surface when trimming the paraffin-block, the paraffin-infiltration was insufficient. You can try to improve the ready blocks by melting them again and let them sit for an additional day in molten paraffin. The residual xylen or even ethanol should evaporate and iniltration should be improved. You can prolong this procedure till cutting works.
Is there a reason why you need to process such a large swath of tissue en bloc? Usually you want one of the dimensions to be very minimal given the poor penetrations of formalin - this means 2-3 mm on one of the edges.
The PI requesting the blocks wanted them large. However in the future I will definitely trim them.
Thank you Gudrun, I will try that next time I have this problem.
Thank you everyone for your help!
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