Re: [Histonet] embryo perfusion

From:Gayle Callis

It is possible totally block all endogenous peroxidase/pseudoperoxidase
using Glucose Oxidase method. You did not say what your enzyme IHC methods
was - peroxidase or alkaline phosphatase, I am presuming HRP or you could
do the alk Phos method instead of HRP.   

I will be happy to send (privately) the glucose oxidase method that works
for minimally fixed frozen sections (acetone) and PFA or NBF fixed,
paraffin embedded tissues.  Sectioning a bit thinner, at 5 um may help in
getting rid of endogenous peroxidase with less to quench. 

You always get some shrinkage in paraffin processing (have a publication
buried somewhere on this, 25% shrinkage seemed to be normal) - it happens
with ALL tissues, delicate embryos often need special schedules to optimize
water removal, clearing  and paraffin infiltration.  It could be that you
are overexposing the fixed embryos to alcohols. This can be adjusted for
less time to avoid removal of bound water on tissue components rather than
just free water found in tissue spaces.  Too long in alcohols, xylene,
paraffin is too hot and even adding heat to each step of processing of
murine tissues contributes to "overprocessing", rather overexposure to
alcohols. It very well may NOT be further fixation BY alcohol, but the
bound water removal problem.  This also leads to poor sectioning, generally
rather dry/friable at times.  

You did not give the processing schedule for these embryos???  


At 02:33 PM 10/2/2003 +0200, you wrote:
>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 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
>Histonet mailing list
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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