RE: Mouse Embryos

From:"Saby, Joseph" <>


I have done some work with similar embryos.  I don't think that 24 hours
formalin fixation is anywhere near long enough to fix embryos of this size,
even with the abdomen opened.  By transferring to 70% ethanol after 24 hours
your are initiating alcohol fixation, which in itself will show in the

When I processed these embryos I separated the heads from the bodies and
submitted in 4 cross sections, then split the bodies into left and right
halves.  I processed the tissues on a non-rodent program, which slightly
over-processed but which had more acceptable results than a rodent program.
I step sectioned through all sections.  I never had a burning problem.

I would suggest getting a temperature recording apparatus which will
continuously record temperature over time.  Place the thermometer in the
oven (the wire lead will not let you place it in the retort) and start a
processing program with no tissues.  This will show if there are any
temperature "spikes" during your processor run.

Good luck!

Joseph A. Saby, BA, HT(ASCP)
Drug Safety Evaluation
Pfizer Global Research and Development
2800 Plymouth Road
Ann Arbor, MI 48105
Phone: (734)-622-3631
FAX:   (734)-622-3866

-----Original Message-----
From: Histo-Scientific Research Laboratories
Sent: Tuesday, April 24, 2001 8:25 AM
To: Linda McGraf
Subject: Mouse Embryos

Dear Histonetters,

We are having burning problems with our mouse embryos.  They are neonates
that are about two cm long and about 1 cm wide- so they are fairly large
embryos.  They are fixed in 10%NBF for 24 hours then stored in 70% ETOH.
They have been cut down the belly for proper fixation.  We are using a
Tissue Tek, VIP  and our processing schedule is: 2  70% for 40 mins each, 1
80% for 40 min. 2 95% for 40 mins each, 3 100% for 40 mins each, 2 xylenes
40 mins each (no heat, PVC cycle on for all alcohols and xylenes), 4
paraffin changes (paraplast, 56 degree melting point) for 30 mins each on 60
degrees.  We have talked with both Bel-Air and Sakura to see if it was the
processor itself, and we have ruled that out.  We change all of our reagents
every 5 cycles- once per week.  We also do a daily clean which consists of
discarding the first 70%, moving #2 up and replacing, discarding the first
100%, moving #2, and #3 up and replacing and the first paraffin and
replacing with new.  No matter how fresh the reagents, we are still having
severe shrinkage and burning of these embryos.  Our other tissues are fine.
Could anyone tell me what we may be doing wrong?  All thoughts are greatly


Tom Galati
137 South Main Street
Woodstock, VA  22664
fax: (540)459-8217

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