[Histonet] Re: Immunohistochemistry with whole mouse embryos
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
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)
>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?
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-4303 (FAX)
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