[Histonet] Mouse pups and processing

[Derek Papalegis]

Pamela Marcum wrote:
>
>
> I have been struggling with this for a while and need some help.  We 
> currently have a project with 0 day mouse pups that are allowed to be 
> born normally and then sacrificed with CO2.  We had several groups 
> earlier that were sacrificed a different way and they processed and 
> sectioned beautifully.
>
> These don't seem to fix well, dehydrate, clear or infiltrate worth a 
> darn.  Since this is my first time using CO2 for sacrifice I need to 
> find out if it causes a problem or if I am just losing it.  I have not 
> ever had this problem before and even re-processing does not help.  I 
> know they are not infiltrating as they are floating in paraffin at the 
> end if I remove them from the processor to a vat of paraffin.  They 
> will not sink.  The pups are 1.2 to 1.3 grams each.
>
> If you can suggest a better way to sacrifice them please let me know.  
> Killing the mother and perfusing her is not an option as these are not 
> our mice.  They are being given as favor so I am limited to some extent.
>
> This was an overnight process with slightly altered alcohols to 45 
> minutes each, 1 xylene at 30 minutes and 2 Shandon Xylene Substitues 
> at 10 hour each to 4 paraffins at 45 X2 and 1 hour X2.
>
> Best Regards,
>
> Pamela A Marcum
> Manager, Histology Special Procedures
> University of Pennsylvania
> School of Veterinary Medicine
> R.S. Reynolds Jr.  CORL
> New Bolton Center
> 382 West Street Road
> Kennett Square, PA 19348
>
> Phone - 610-925-6278
> Fax     - 610-925-8120
> E-mail - pmarcum <@t> vet.upenn.edu
>
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Hi Pamela,
I would recommend using decapitation as your euthanasia method instead 
of CO2. Use Bouin's to fix instead of formalin. I usually decapitate and 
then slightly cut the abdomen with a razor blade to help the fixative 
penetrate. I have left pups in Bouin's up to 48 hours with no adverse 
affects.How you proceed really depends on what sections you want. Most 
investigators I have encountered initially want longitudinal sections 
cut but they soon realize that this does not yield any useful 
information from the slide. Longitudinal sections "look pretty" on the 
slide but they are practically useless to demonstrate all the organs. 
The best way to make sections of pups is to take 5-7 cross sections 
after it is completely fixed. I have a diagram of a pup with lines 
through it showing me where to take my sections from. This ensures 
consistency from pup to pup. I just process the pups on my normal 
processing schedule and have had great results from this.

Feel free to email me if you have any questions about this or would like 
me to send you the pup diagram of where to take sections from.

Good luck

Derek

-- 
Derek Papalegis HT (ASCP)
Histotechnician
Division of Laboratory Animal Medicine
Tufts University 
136 Harrison Avenue
Boston, MA 02111
phone: 617 636-2971
fax: 617 636-8354