[Histonet] hand processing schedule late mouse embryos

[Nicole Collette]

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