[Histonet] Paraffin processing late stage mouse embryos

Tue Apr 19 18:31:36 CDT 2005

Teri,

I do a lot of work with mouse tissue and I find that if the tissue is
blowing up on the water bath that most likely there is inadequate paraffin
infiltration, which may be a result of inadequate fixation. If the staining
you are doing can stand a longer time in fixative I would recommend a week
in NBF making sure you have an adequate volume. I would try a gentler
processing schedule. 70%ETOH 3 hours, 80% ETOH 1 hour, 95% ETOH 2 changes 20
minutes each, 100% ETOH 2 changes each, Propar 3 changes 30 min each, 4
paraffin 4 changes 30 minutes each. I personally prefer Propar to Xylene for
mouse tissue and four paraffin baths. Hope this is helpful.
c

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317

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-----Original Message-----
From: Johnson, Teri [mailto:TJJ <@t> Stowers-Institute.org] 
Sent: Monday, April 18, 2005 12:48 PM
To: Histonet
Subject: [Histonet] Paraffin processing late stage mouse embryos

Is anybody doing paraffin processing on E18.5 - P0 mouse embryos that
can help us optimize our in-house protocol?  I know that when possible
one should bisect embryos for best fixation and processing.
Unfortunately we can't always do that.  In some instances, we are able
to bisect the samples just posterior to the forelimbs and process only
the top 1/2 of the embryo.  Even after poking some holes and immersion
for 72 hours in 10% NBF, combined with a rigorous processing schedule (2
hours/station with a typical automated tissue processor setup--same
schedule as we use for decalcified whole adult mouse heads!), our
samples just aren't sectioning well, and they tend to blow up on the
water bath.  A test with samples without holes poked in them vs. ones
with holes produced no discernable improvement in processing.

A google search turned up one lab (evidently hand processing) who
recommends removing the skin of this size embryo (E15.5 - E18.5)  and
removing the head and/or limbs if not needed for study.  Fixation and
processing: fix 1 hour @ 40 degrees C, change fixative, then 40 degrees
C overnight. The following are done at room temp or refrigerated for IHC
or ISH: dehydrating in 30% ETOH 1-2 hrs, 50% ETOH 1-2 hrs, 70% ETOH 4
hrs or O/N, 95% ETOH 3 hrs x 2 or O/N, and 100% ETOH 1 hr x 2.  Embryos
can be stored at -20 degrees C at this point.  Carrying on with
dehydration and infiltration: 100% ETOH 1 hr x 3 @ RT, Xylene 30-40 min
x 2 (check embryos*), paraffin 40 minutes x 3, paraffin embedding.

*No indication of what we're actually checking on these samples

I do think that removing the skin on these guys would help immensely.  I
also think that additional dissection when possible is critical to good
fixation and processing.

Any comments on the above processing schedule?  Is anybody doing these
later stage mouse embryo samples using a protocol that's working for
you?

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri  64110
tjj <@t> stowers-institute.org

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