Suggestions RE: [Histonet] Re: a basic question about immunohistochemistry

[gayle callis]

Hopefully, your next study using rats will have more than one animal.  My
primary research contact and his post docs along with several other research
groups in our department work with rodents.  Their studies are always set up
with more than one animal, even pilot studies.  You need to have good
correlation of results and a single animal simply does not provide that
correlation. Consequently, we have anywhere from 5 to 6 animals or more in
any particular treatment group and the same for a control group. These
animals are kept in a controlled environment animal facility during
experiments under auspices of federally mandated guidelines one is required
to follow or be in BIG trouble.     

One animal is NOT a good way to get results especially when the animal dies
before a euthanasia/time endpoint for tissue collection and you need
perfusion fixation as part of your protocol.  This animal has fallen out of
your research parameters and statistically not useful.  We do not trust any
IHC results from deceased animals and do not do immunostaining when this
happens.  When an animal has been deceased for many hours (and how would you
know!), we either discard or necropsy to determine cause of death especially
if infection is suspected. If more than one or more animal dies, then
finding cause of death IS important before repeating the experiment if
possible either overdosing of some drug, chemical agent or massive infection
from infectious agents are involved in the study. We do document if an
animal dies during an experiment as mortality may be an important statistic
to note.   

IHC will more than likely be very skewed, with untrustworthy results, due to
post mortem tissue autolysis affecting antigens especially if the animal has
been deceased for many hours.  Necropsied tissues will show autolysis too.
If you use deceased experimental animal results when you need tissues
correctly fixed from a euthanized animal for any manuscript destined for
publication, reviewers will more than likely frown upon what you did, and
reject the manuscript.   

You need to repeat your experiment with more animals, and also have some
type of sham treatment control animal group.  

Bob Richmond made a good point - I would take John's comments to
heart..................journal manuscript reviewers and thesis defense
graduate committees can be much tougher than John is!!!  
Gayle Callis
HTL/HT/MT(ASCP)
Bozeman MT 


  

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Robert
Richmond
Sent: Sunday, November 29, 2009 11:35 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Re: a basic question about immunohistochemistry

Thomas Jasper certainly points out an issue of politeness: >>Being
chastised on this list and calling your work "bad science" is totally
out of line and certainly does not help you out.<<

I agree up to a point - but frankly, if John Kiernan called my work
"bad science" I would seriously reconsider what I was doing.

(Which on occasion he has, come to think of it.)

Bob Richmond
Samurai Pathologist
Knoxville TN

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