[Histonet] Two potential problems here Re:confocal microscopy

[Gayle Callis]

First, if you fix with PFA, you need to cryoprotect the prefixed tissues 
with 25 to 30% sucrose overnight or longer (tissue should sink to bottom of 
solution) or you will have poor frozen sections.  Just rinsing with PBS is 
still keeping high water concentration in the tissues for water ice 
formation problems.

Secondly, if you snap freeze OCT embedded embryos without fixation, in 
other words fresh embryos, you will eliminate increased autofluorescence 
problems created by paraformaldehyde fixation.

If they are already fixed, then you can still cryoprotect, and do snap 
freezing, cryosectioning or you can try paraffin 
embedding.  Autofluorescence may be present at a level you can work with or 
deal with autofluorescence by using a fluorophore that is a contrasting 
color i.e. red rather than yellowish-green.  Alexa 555 from Molecular 
Probes can be found conjugated to secondary antibodies and 
Strepavidin.   Alexa fluorophores are more stable, less photobleaching when 
using Confocal or CLSM microscopes.



At 09:35 AM 3/9/2005, you wrote:
>RT,
>The reason frozen sectioning is recommended for CF is that flurescent
>labels are often used and usually work best on frozens, you can rinse the
>PFA from your fixed hearts with PBS or Tris buffer and then try freezing
>them to make sections for IHC.
>Patsy
> > Hi, I have fixed some E13.5 and E16.5 mouse hearts in 4% PFA O/N and
> > now i am thinking of doing confocal microscopy. What is the best way to
> > embed? I see in the literature that cryo sections are better, since i
> > already fixed them what would be the best bet?
> > Thanks
> > RT
> >
> >
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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)