not a strange request on mouse aorta cultures

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From:Gayle Callis <>
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Have done this, they wanted to see the vascular (actually angiogenisis)
development off the aortic ring, and the fibrin and collagen gel really did
not interfere with the morphology of the ring or the vessels that grew off
of it.

We fixed our rings, intact on the culture plate, basically fixing them in
place in the gel contained in round culture dishes, using METHCARN, methyl
alcohol/Carnoys, or the fixative of choice.  We did not put chloroform in
the fixative, too toxic, and not needed, no fat removal involved.   

The fixed tissue/gel was then gently lifted off after cutting around the
ring with a scalpel blade, to not disrupt tiny vessels (these can be single
cell thickness).
We rinsed with absolute alcohol, and did not process any extra time with
this, just started the VIP at 100% station.

Processing for these MethCarn fixed rings was start in 100% alcohol, xylene
or sub 2 changes at 30 min each, 4 changes of paraffin 30 min each, total
time was 3 hr 15 min.
Shorter processing is a good idea, the gels can become a tidge hardened.  

Cutting was serial at 5 um, starting right from the first hint of being in
the block, to not miss the tiny vessels, then proceeding through the block,
the ring was cut in crosssection to show the lumen and vessels growing off
the edges.  

We did Massons trichrome, H&E, and Factor VIII Dako 1:200, purified,
without any antigen retrieval, and had interesting, nice staining.  The
collagen/fibrin gels don't take up too much stain, as the cells/tissue are
sufficiently different to identify.

Reference for you is:

Nicosia, RF and Ottinetti A.  Modulation of microvascular growth and
morphogenesis by reconstituted basement membrane gel in three dimensional
cultures of rat aorta: a comparative study of angiogenesis in matrigel,
collagen, fibrin, and plasma clot

In Vitro Cell Dev Biol. 26:119-128,FEb 1990

This group did NBF the GMA embedment, but we did not want to mess with this
plastic and immuno, nor antigen retrieval.  NBF should work fine, and one
could use the regular Carnoys without chloroform or Paraformaldehyde.


Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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