[Histonet] Re: Immunohistochemistry with whole mouse embryos

From:Gayle Callis

Prolong gold antifade ready to use, but is this going to flatten the embryo 
under a coverslip?   If the CLSM is fitted with an inverted microscope, 
there are special petri dishes that have coverslip bottoms.  You can float 
a stained embryos in PBS without having to use mounting media over the top 
of the embryos to prevent drying.  It does not have to be antifade mounting 
media - but you can try both for scanning and z stack imaging.

Personally, I prefer Alexa dyes for CLSM - they are brighter and do NOT 
photobleach as readily.  Check out Molecular Probes Alexa Fluor selections 
on line.   Some are going to far red fluorophores to avoid autofluorescence 
issues,- I suggest you look into that also.

Permeabilizing can be done with a detergent, Tween 20 or Triton X 100, and 
with the thicker embryos, you may need to stain overnight at 4C to have 
adequate penetration of everything.  I have seen this in the literature 
(methods and materials for both brain and embryo work).  I am not sure of 
the concentrations.

At 08:03 AM 4/5/2006, you wrote:
>I am having problems to perform some whole mount stainings protocols in 
>mouse embryos E10.5:
>1) The whole mount staining protocol for PECAM (anti-mouse PECAM 
>monoclonal antibody MEC13.3-Pharmigen) with mouse embryos E10.5 (10dpc). 
>The color reaction (using DAB takes 10 min and the embryos are all black) 
>however, after
>pos-developing washes I don't have any signal. I think is a question of 
>penetrance of the Antibody. Do you have some tips to permeabilize the embryos?
>2) If I want to do an immunofluorescence protocol with whole mount mouse 
>embryos E10.5 (using a Cy3 conjugated monoclonal Antibody - red spectrum, 
>Absorbance:552nm Emission:570nm) and I want to analyse the whole specimen under
>a confocal microscope. What should be the mounting medium more appropriated?

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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