Re: Whole embryo IHC
|From:||Barry Rittman <email@example.com>|
One thing that might work for you is storage in 50% glycerol. The embryos
are relatively small. The glycerol solution is gradually added to
increase its concentration and prevent collapse of the embryo that would
occur if placed directly in 50% glycerol. This can be accomplished either
using graded glycerol solutions or with the embryo in saline, adding
glycerol dropwise to attain the desired concentration.
This is just a suggestion, I have never tried glycerol with DAB stained
specimens but you can do a quick trial run. I do know however that this
concentration of glycerol prevents growth of bacteria and viruses. Have
used it succesfully to store specimens for many years.
At 01:41 PM 07/16/2001 -0700, you wrote:
Currently I'm involved with a project doing immunohistochemical
staining of whole (not sections) mouse embryos staged at e9.5-e10.5.
Although I'm obtaining a good intense staining pattern localized to
the site of interest with minimal background, I'm unable to retain it
for long term purposes. The protocol is a two step indirect,
monoclonal primary followed by a peroxidase conjugated IgG F(ab)2
secondary and using either DAB or DAB/NiCl as my substrate. After
development of the substrate to desired intensity, I placed it in
0.1M Tris pH7.4 with 0.5% Triton X-100 to stop the reaction. Photos
are taken at this point and then it is transferred to one of the
following solutions: PBS, 10%NBF or 4% paraformaldehye at 4C. After
1-2 days there is significant loss of staining intensity although
what remains is localized to site of interest, while after a week of
storage the signal is so weak that I cannot really use it at this
So my question is: What can I store these stained embryos in to
stabilize the staining and store them for long term purposes
(possibly for months).
I'm using someone elses computer to send this off this message but
any responses can either be posted directly on the histonet or sent
directly to me at: firstname.lastname@example.org.
Thanks in advance for any respons
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