[Histonet] Re: Mouse pups and processing

Sun Feb 10 07:57:55 CST 2008

Thank you Amos and I can appreciate your problem.  Your discription of sqishy
is
prefect.  I have also referred to the outer skin as lacy in appearance when
attempting to section it.  The other point I would make is the brain is mush
with no form as if it is autolysized totally.  

I have tried splitting the abdomen and it does not help or is so minimal it
doesn't help much.  I have tried changing the processor and always process
other tissues at the same time to show the PI it is not the reagents and
paraffin.  We have processed other pups before this over the last year and know
they were not killed with CO2.  They work great and section great (I still
split
the abdomen).

I am at a loss and finally turned to all of you on HistoNet help.  You are
great
and even though we may not have the answer yet we will I am sure of it. I know
this is an ongoing problem for researchers with pups.  Maybe with this CO2
thing being a popular way for sacrificing animals we have hit on something that
happens in pups and not adults.  I had not really ever worried about it until
now as I did my own sacrifice and it was fine.  I wish they weren't so small I
would attempt prefusion with a 2cc syringe and blunt needle as a last resort. 

I want to THANK everyone who has and will take the time to answer and help me
with this issue.  At least I can show the PI our lab is not the only one with
the problem and we are all attempting to find a way out of it.  I think from
all I have seen we should be more aware of how the pups are sacrificed and note
any differences between the way our PI's sacrifice them.  Maybe all of the
material submitted to the Histology area for research on pups and other animals
should be recorded and we might see differences they don't in method.
Recommendations from IACOCC do not take our area of histology into
consideration as they are controlling the most humane and painless method of
sacrificing animals over all.  Perhaps one of us can get a paper out of this or
a talk in the future that helps everyone.   

Who knows I even checked the phase of the moon this last time and it did not
help.

Thanks again and still looking.  If I solve this you will all be teh first to
know.

Pam

Quoting Amos Brooks <amosbrooks <@t> gmail.com>:

> Pam,
>      We were having a similar problem with one of our researcher's P0 mice.
> I don't know how they sacrificed them, but they too dod not process at all.
> They were squishy!
>      I reccommended they either bisect or at least make a good midline
> incision prior to fixation. It still isn't perfect (is it ever?), but it
> seems to be a bit better. This allows better solution penetration. Give it a
> whirl and let us know how it goes.
> 
> Amos
> 
> Message: 13
> Date: Fri, 08 Feb 2008 15:52:34 -0500
> From: Pamela Marcum <pmarcum <@t> vet.upenn.edu>
> Subject: [Histonet] Mouse pups and processing
> To: Histonet <@t> lists.utsouthwestern.edu
> Message-ID: <6.2.5.6.2.20080208154217.01c8a9b8 <@t> vet.upenn.edu>
> Content-Type: text/plain; charset="us-ascii"; format=flowed
> 
> 
> 
> 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
> 