Re: Mouse Embryos

From:Gayle Callis <>

Neonates may need longer fixation, these are good sized - 24 hours is not a
long time, make sure you have adequate fixative on the specimenm, 20 to 1
ratio OR alcohols during processing will finish the fixation for you.  A
dessicating, shrinking experience.  You would know if you test a neonate,
fix your way, reopen the whole body to look for reddish unfixed tissues,
extend time if needed. 

By burning, what do you mean????  Describe this please.  Do you dry slides
at 56 - 60C for 45 min - 1 hour, if you use higher temps, you could be
burning the tissues, this could be source of this problem.   We air dry
neonate sections overnight at RT (on plus charge slides) then go to 58C
oven for 30 min.  No burned or shrunken sections.  Our schedule is same as
yours for alcohols, etc, but 1 hour per station, even in paraffins.    

***Do you mean you take the 80% and move it into the 70%?? and rotating a
95% into an 80%?   If so you are NOT replacing that 70% with a 70%, if so
you are actually raising the concentration of alcohol by putting the 80%
into this slot.    You get some water carryover, but bet it is not a 10%
difference to make the 80% turn into 70%??!!!   Knew a gal who did this,
her tissues weren't looking too great after some major overdehydration!!!
Her idea was to save money and probably did it at the expense of bad tissue

70% should be replaced with 70%, same for 80%;   95% and 100% can be
rotated down.  Your problem, if you rotate as I described at *** could make
neonates overdehydrated, and look dessicated, or 'burned'?  Not sure what
this means   

Another thing to do is a dummy run during the day, start processor and
watch temperature changes, at each pump in and processing time (set it for
30 min per station) observe the temperature to see if it goes up.   If the
processor has a temperature problem, you have better argument with Sakura.
You can do this with the alcohol stations so you can abort the run before
it goes into xylene or paraffin.  For any temp changes on paraffin, make
sure you see the last station  at the end of a regular run, check temp to
see what that is.  Program to have your run end when you are present to
observe it.  Best way to check the temp is OPEN up retort and use a
thermometer, a readout could be faulty, and you need to test temp by
immersion of thermometer in paraffin while it is in the retort. Same for

We had to do this some years ago, won the battle, and Sakura graciously
corrected the problem. 

On a daily clean, replacing 70% is good practice, but I don't know if you
have to replace or rotate so frequently, must get expensive??  If you are
not running a ton of tissues daily, you probably only need to rotate every
other day or so and maybe not at all.  However, new 70% is a good idea to
get rid of residual NBF that ppts out in higher concentrations of alcohol.
However, this is a call based on number of tissues put through daily,
different for every lab.        

Enough of Dear Ms Blabby

08:24 AM 4/24/01 -0400, you wrote:
>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
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)

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