[Histonet] embryo perfusion



Need help on PFA perfusion techniques on rat and mice embryos (E18). I've
tried perfusing (intracardiaque) the mothers (10 minutes with PBS/heparine
then 30 minutes with PFA (pump speed of 2ml /min for mice and 25ml/min for
rats), spread the babies out on the table...one sees that the saline
solution does clear the blood, and the PFA does harden them up. I then make
a sagital slice through each embryo and post fix for 24h followed by
paraffin embedding. I then make 7µ  sagittal slices.  But in the stained
sections there are still an abundance of red blood cells that are a big
source of background for IHC, and the tissue seems underfixed because there
is shrinkage (alcohol fixation during embedding). I've heard that one can
perfuse individual embryos via the umbelical cord. Does anyone have a
description of this with a schema or explanation on how to find the right
artery, pump speed and PFA volume.

thanks for your time,
Nancy Walker
Molecular Biology Scientist

Sanofi-Synthelbo Research
B.P. 37 Labége Innopole

tel : (33)561004179  fax :(33)561004001

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