[Histonet] Cryosectioning versus paraffin sectioning mouse embryo

[Gayle Callis]

The difference between cryosectioning and paraffin sectioning needs to be 
clarified.

For cryosectioning, you can perfuse a mouse (not sure how this works with 
pregnant mouse, or do immersion fixation of collected embryos, then 
cryoprotect with 25 - 30% sucrose overnight, SNAP FREEZE with extreme cold, 
there have been methods discussed at length on Histonet, then cut sections 
in a cryostat or cryosectioning.  You can also snap freeze fresh, unfixed 
tissue and do fixation in 4% PFA after the section is cut and mounted on a 
slide.

Paraffin sectioning is taking a fixed embryo, processing it through alcohol 
gradient, clearing alcohol, and infiltration with paraffin.  This 
sectioning is done on a microtome and IS not cryosectioning.  Paraffin 
would have to be removed from sections prior to staining.

You either do one method or the other. but paraffin sections are NOT cut in 
a cryostat.

There are some websites on mouse brain, go to www.mbl.org for mouse brain 
library.

Mouse embryo 
atlas/photo    http://embryo.soad.umich.edu/animal/cdAtlas/cdAtlas.html
Embryo, mouse MRI             http://www.mouseatlas.caltech.edu/home.html
Embryo stage selector         http://genex.hgu.mrc.ac.uk/Atlas/intro.html
High Resolution Mouse Brain 
Atlas:      http://www.hms.harvard.edu/research/brain/goal.html#top

These should at least teach some brain anatomy, and figure our how you want 
to orient the tissue for final sectioning and the view you need to have.


I am going to start processing mouse embryos for in situ and
immunohistochemistry, and since I am a novice, I have a few very basic
questions that I am hoping to find answers to!  First, I was wondering
if it is absolutely necessary to embed embryos in paraffin for
cryosectioning.  I usually work with brain tissue from adult mice, and I
never have to embed in paraffin -- I always freeze in OCT.  In this
case, I will be sectioning embryos at ED14.5 and ED17.5.  Also, I was
wondering about a simple fixation procedure for 'older' embryos.  Is it
feasible for fixation to involve only fixing cryosections in 4%
paraformaldehyde?  Lastly, if my goal is to look at expression patterns
in the embryonic brain, what would be the best approach/angle to
sectioning?  Specifically, I am interested in having a good view of the
ventricular zone and dentate gyrus.

I would greatly appreciate any and all advice on these matters!  Thank you
in advance!

Ann


Gayle Callis
MT,HT,HTL(ASCP)
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)