[Histonet] hand processing schedule late mouse embryos

[Rene J Buesa]

Under separate cover I am sending a protocol that will work well with your subjects.
Since you are doing manual processing you can easily use the reagents I recommend and eliminate xylene and its terpene and alkane based substitutes.
René J.

--- On Fri, 2/13/09, Nicole Collette <collette2 <@t> mail.llnl.gov> wrote:

From: Nicole Collette <collette2 <@t> mail.llnl.gov>
Subject: [Histonet] hand processing schedule late mouse embryos
To: histonet <@t> lists.utsouthwestern.edu
Date: Friday, February 13, 2009, 1:25 PM

HI, All,

One project finished, another just beginning. I am about to embark on a journey
into the land of immunohistochemistry, with late mouse embryos E14.5, E16.5, P0
to examine bone markers in conjunction with LacZ and/or GFP.

We have sadly lost our cryostat (so IHC for the GFP on paraffin sections), and
our tissue processor - both belonged to a friendly investigator down the hall
who has moved on. So, I am processing by hand.

For hand-processing, I have had to do some rigging, and I do the wax steps in a
hyb oven to try to keep the wax (TissuePrep, Fisher) at around 63-65C, while
trying my best to keep the molds, cassettes, and tools with as few giant globs
of solidifying wax as possible. As a result of using the hyb oven, we are forced
to use Clearene (D-limonene), --or some other xylene substitute that could be
recommended--,  instead of xylene for the processing. If anyone has a
recommendation for a better alternative there (aside from a tissue processor
which will have to wait at least until the next grant gets funded--oooh, unless
someone has an old one they want to donate, preferably table-top), I'm all
ears.

My schedule was given to me by a friend who does cartilage, no older than
E14.5, and are basically half hour steps for each ethanol, half hour steps for 3
wax steps at the end. Will this be enough time for infiltration of older samples
without vacuum? Should I increase my steps to 1 hour for these older embryos? I
am optimizing my fixation at 1hour/mm thickness, with the embryos skinned (4%
paraformaldehyde in PBS, I decided to start here since I don't yet know much
about the problems I might encounter with a particular antigen). I have tried
the 30 minute schedule with adult decalcified bones and have not had fantastic
sections. I suspect it could be incomplete washing of the EDTA before
infiltration, but it's possible that the processing schedule is just not
long enough. Any advice?

Thanks in advance! Happy Friday!

Sincerely,
Nicole Collette
Lawrence Livermore National Lab/ UC Berkeley

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