I think Dereks information regarding decapitation is very good and I don't have much to say about fixation and processing in addition to his but I would say sit with your IACUC committee immediately before you do any more euthanasia or accept anything from someone who hasn't gone through IACUC. Euthanasia procedures for such pups are completely different than for adults because of their resistance to hypoxia and the CO2 euthanasia. Every committee follows (or should follow) rigid standards for the welfare of animals being sacraficed and can differ in things such as length of time, cycles in CO2, paper in the chamber, secondary euthanasia techniques, who can do this and who can't. I've seen pups come out of CO2 chambers for extended times and still be alive and start kicking due to their built in CO2 resistence. I think it is our moral duty to see that animals suffer as absolutely little as is possible and that is why IACUC committee's exist and their regulations rigid.
-------------- Original message --------------
From: 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 - email@example.com
> > _______________________________________________
> > Histonet mailing list
> > Histonet@lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 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 Papalegis HT (ASCP)
> Division of Laboratory Animal Medicine
> Tufts University
> 136 Harrison Avenue
> Boston, MA 02111
> phone: 617 636-2971
> fax: 617 636-8354
> Histonet mailing list
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