Dear Tom

in our lab we have recently tested the paraffinization of normal and
knockout mouse embryos starting from developmental stages ES12.5 up to P1.
Our experience are as follows:

Embryos from ES 12.5, 14.5, 16.5, 18.5, 19.5 and P1 129sv mice were fixed
in 4% phosphate buffered formaline for 24h at 4°C. Of each developmental
timepoint some animals were skinned (removal of skin over the belly and the
back), whereas some were left untreated prior to paraffinization.
Paraffinization was carried out using either a short programm (12h) or a
long paraffinization program (24h). The baths were heated 37°C for alcohol
and xylenes and 58°C for paraffin. Thereafter, embryos were embedded in
paraffin, 5um sections were cut and H&E stained.

Up to ES16.5 paraffinization was achieved without any problem regardless of
skinning or program used. But for all older embryos we experienced what you
have described in your email: massive shrinkage of the embryo including
head and body and a burnt-like aspect (darkbrown color). In addition, the
embryos were soft and juicy and smelled not very pleasent (xylenelike). In
addition, I was not able to embed them as the embryos did not at all
combine with the paraffin. I left them over night and they dried completely
thereby separating from the paraffin. Cutting was impossible.

In a second set of experiments we cut embryos from ES 18.5, 19.5 and P1
either in half sagittaly (midline) or we cut the head of and the body in
two sections (transversal).
Paraffinization of the midline sectioned embryos yielded in good
penetration and nice histological cuts.
In contrast, the whole head alone was shrunken as beforehand and the whole
body was shrunken as well only if the transversal cut was made above the
diaphragm.
Again, we tested to cut the head in half either sagittaly or coronally, and
we cut the body transversally paying attention that we cut below the
diaphragm. This time all body parts have been well preserved and nice cuts
could be obtained.

I assume that the development of the skin and its underlying tissue layers
and the development of cartilage in the skull prevent efficient penetration
with paraffin. My impression was that fixation, dehydration and clearing
work well with whole mount embryos. But paraffinization is prevented after
ES 16.5 due to skin and cartilage development.

So, I hope that this short summary of our experiments is of any help to
you. In addition, I hope that some colleagues from the histonet working in
this area will be stimulated to comment on this. I have to admit that I was
not very lucky to find state of the art protocols in the literature. Even
the book from Kaufman, dealing with mouse embryology and showing beautiful
pictures, did not accuratelly describe the technical procedure.

Kind regards.

Michael




Michael Stumm, MD, Lab 405
Department of Research
Kantonsspital Basel
Hebelstrasse 20
4031 Basel
Switzerland
phone: ++41/61/265 23 87
fax: ++41/61/265 23 50
email: michael.stumm@unibas.ch