[Histonet] Paraffin embedded mouse embryos - too dry

[Barbara Schormair]

Dear all,

I'm having trouble with my paraffin embedded mouse embryos. We use E12.5 
to E15.5 embryos and we embed and cut the whole embryo. When we try to 
cut, the paraffin sheets are fine, but as soon as we get to the embryo, 
the tissue disrupts and tears. I also hear rasping or scraping sounds 
when cutting throught the embryo, so the problem seems to be that the 
tissue is too dry or too hard.
Here is my current protocol:
Dissect embryos from the uterus and amnion in 1x PBS, then fix overnight 
at 4°C in 4% PFA. Transfer to 70% Ethanol and store at 4°C.
After 2 days to several weeks, we continue with the processing as follows:
E12.5: 96% Ethanol for 15min, 100% Ethanol for 15min, Xylol for 15min (I 
also tried 10min Xylol and 8min Xylol, but with the same results).
E15.5: 96% Ethanol for 30min, 100% Ethanol for 30min, Xylol for 30min (I 
also tried 20min Xylol and 15min Xylol, but with the same results).
Then bring into Paraffin and leave overnight at 65°C, next day embedding 
and cooling down on-5°C cooling plate. Store at 4°C.

What is the problem? Storage in 70% Ethanol for too long? Xylol 
incubation times too long or too short?
What is the critical step - the ethanol incubation or the xylol 
incubation - should I try different times in Xylol or also try and 
change the Ethanol incubation times?

Thanks in advance for any help!

Best regards,

Barbara



-- 
Dipl. Biol. Barbara Schormair
PhD student 
Institute of Human Genetics
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e-Mail: barbara.schormair <@t> helmholtz-muenchen.de

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