I would agree that one needs to check with the IUCUC group. They were unhappy with our using CO2 and recomended ether. We now etherise them and then decapitate them. jane
"Life is short - make haste to be kind"
Dr. Jane Flinn
Director, Biopsychology Program
George Mason University, 3F5
4400 University Dr.
Fairfax, VA 22030
Phone: 703-993-4107
Fax: 703-993-1359
----- Original Message -----
From: patsy ruegg
Date: Saturday, February 9, 2008 12:11 pm
Subject: RE: [Histonet] Mouse pups and processing
> I agree decapitation is the way to go with pups, that way you can
> get them
> fixed quickly. We even dissect the heads longitudinally in the middle
> between the eyes down the middle of the nose because we are
> interested in
> sinus development. After fixation in NBF for 24 hrs. I process
> these on a
> regular 1 hour per reagent tissue processing schedule, they cut
> and look
> really good.
> Patsy
>
> Patsy Ruegg, HT(ASCP)QIHC
> IHCtech
> 12635 Montview Blvd. #216
> Aurora, CO 80010
> 720-859-4060
> fax 720-859-4110
> pruegg@ihctech.net
> www.ihctech.net
>
> -----Original Message-----
> From: histonet-bounces@lists.utsouthwestern.edu
> [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of
> koellingr@comcast.net
> Sent: Friday, February 08, 2008 4:14 PM
> To: Derek Papalegis; Pamela Marcum
> Cc: Histonet@lists.utsouthwestern.edu
> Subject: Re: [Histonet] Mouse pups and processing
>
> Pam,
> 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.
> Ray
>
> -------------- 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 - pmarcum@vet.upenn.edu
> > >
> > > _______________________________________________
> > > 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
> >
> > --
> > 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
> >
> >
> > _______________________________________________
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> > Histonet@lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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