[Histonet] rat perfusion
mcauliff <@t> umdnj.edu
Wed Dec 19 09:58:27 CST 2007
Your first problem is cold perfusate.Why?? All this does is
constrict the blood vessels and imped fixation. The concept that cold
fixative slows down autolysis and improves fixation is a myth What
really happens is that the reaction of the fix with the tissue is
slowed. Yes, there are some instances when cold fix is better, but this
is not one of them.
There is no need to use so much saline washout; you are using well
over 10X the circulating blood volume. What you are doing is waiting 5
to 10 minutes to begin fixation! You will never wash out all of the
blood, it is not necessary so don't try. Less than 100 ml is plenty. As
soon as the nose blanches,switch to fix. Room temperature fix, not cold!
Use a 15 or 16 gauge needle, the others are too small to achieve
adequate flow. When you clamp the heart you may be impeding the flow of
fixative due to misplaced clamp, try holding the needle in place and see
if that improves your results.
I have had perfusions that looked "good" but gave mediocre
morphology and perfusions that looked "bad" with great morphology. I
would expect the 'dance' ~90% of the time.
I would slice the brain for post fixation, unless you must cut
through the whole brain.
I think the tearing problem is due to inadequate fixation. The steps
above should fix (ha!) it.
Neil Fournier wrote:
> I have decided that our lab needs to revise our current protocol for rat intracardiac perfusion b/c the quality of perfused tissue has been reduced significantly over the last few months. I also thought it would be best to call upon the significant expertise of many of the histonet members. I hope that you might have some important suggestions and comments for us. I also apologize a priori for my naivety on this subject.
> Our protocol is as follows (for an adult rat typically 300 to 500 g):
> 1) Animals euthanized with a high dose of Euthanyl.
> 2) Perfuse cold 0.9% physiological saline (on ice). Using a perfusion pump with an 17 or 18 gauge blunted needle inserted into the ascending aorta. (I used to use 15 or 16 gauge needles) The heart is clamped to secure the needle tightly in place and the right atrium cut. Typically we perfuse between 300 and 500 ml over a five to ten minute period.
> 3) Perfuse cold 4% paraformaldehyde (on ice). (e.g., 40 g of paraformaldehyde dissolved in 800 ml warm PBS, filtered, cooled, and pH 7.4 with solution toped to a final volume of 1000 ml. All solutions are made up fresh, usually the day before or at least a few days before perfusion is required. Solutions are discarded if older than one week). The flow rate is 300 and 500 ml over a 10 to 15 minute period.
> *** I have noticed from time to time that when some students are perfusing some of the animals do not exhibit the typical spontaneous movements during fixation. However, sometimes the body will still appear rigid and fixed despite not showing these typical movement responses. I am uncertain if the classic "formalin dance" has to be a characteristic of a good whole body perfusion and was wondering what other people's take on this situation is.
> 4) Brains are removed and postfixed for 48 hrs before being sectioned on a Vibrating microtome at 50 microns. Tissue sections are usually stored in a ethylene glycol based cryoprotectant solution at a temperature between 20 and 25 degree C using a protocol identical to Gloria Hoffman's procedure (Peptides 1986, 7(1): 155-159.)
> Before I developed significant allergies, which makes my contact with the laboratory animals now at a minimal (poetic justice), I never had the same degree of problems that we seem to be observing lately. Although sectioning is not problematic with this tissue, the quality of tissue after IHC staining is generally poor (i.e., significant tearing of the tissue throughout etc.). Tissue is stained using the free-floating method and tissue is transferred from rinse to rinse using Netwells. I realize that this method in itself can contribute to some damage. I am not completely sure what the problem is but the consistency of this problem across animals and multiple experiments suggests to me one possibility is the actual perfusion procedure and that tissue integrity is minimal.
> Any suggestions and comments are welcomed. lastly, I was hoping that someone might know of a good book that discusses perfusion techniques in greater detail. Hopefully with some pictures as this is the often the best way to teach students (including myself).
> Thanks for the help,
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> Histonet <@t> lists.utsouthwestern.edu
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
mcauliff <@t> umdnj.edu
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