RE: [Histonet] embryo perfusion
|From:||"Charles W. Scouten, Ph.D." |
Even neonates, probably embryo's, have a blood pressure comparable to other mammals, up above 100 mm Hg. systolic. 2 ml/s per minute will generate how much pressure? You need to be comparable to physiological pressure or above, or you will not get the red blood cells out. Too far above will damage tissue. I would guess from the rate and results that you are way below physiological pressure, maybe 20 mm Hg. The capillaries won't open and release their blood. Then fixative will not flow through the occupied capillaries.
Try controlling your pressure of perfusion, not your flow rate, unless you know what flow rate will generate what pressure. Use at least 150 mm Hg. To avoid shrinkage, use the Perfusion One system, see the link:
Charles W. Scouten, Ph.D.
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300
FAX 314 522 0377
From: Nancy.Walker@sanofi-synthelabo.com [mailto:Nancy.Walker@sanofi-synthelabo.com]
Sent: Thursday, October 02, 2003 7:33 AM
Subject: [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,
Molecular Biology Scientist
B.P. 37 Labége Innopole
31676 LABEGE CEDEX FRANCE
tel : (33)561004179 fax :(33)561004001
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