Hi Neil,
I agree with Geoff. You don't need that much saline for the flush and
it would be optimal to get the fix in ASAP. Usually I find ~80 ml is
more than sufficient to flush and and if the heart was still beating
when the perfusion begins, that is more than sufficient. I have also
used room temperature saline, followed by 300-400ml 4% PFA also at room
temperature. In most perfusion I would see a dance but not all. I
would also ask the students to check that position of the needle is
still correct, ie in the ascending aorta shortly after the start
perfusing with saline, I have found from personal experience, that
sometimes the needle moves while they clamp it in place.
Another question: you are using netwells for the rinses but what about
the incubations? Are you transferring sections from incubation wells to
netwells? That can also be a point of damage. Just make sure they are
really careful with the transferring if you are. I have seen beautiful
sections get torn up from transferring and also sections from the same
rat, handled by two different individuals come out looking very different.
Have you tried staining a slice immediately after vibratome sectioning
without immunohistochemical processing to see if the morphology is ok?
Just some thoughts ... good luck with trouble shooting.
Angelina
> Hi Neil:
>
> 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.
>
> Geoff
>
> 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,
>>
>> N
>>
>>
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>>
>>
>>
>
>
--
~~o~~o~~o~~o~~o~~o~~o~~o~~o~~o
Angelina Y. Fong, Ph.D.
Department of Zoology
Biological Sciences Building
6270 University Boulevard
University of British Columbia
Vancouver, BC, V6T 1Z4
Canada
Ph: (604) 822-5990
Fax: (604) 822-2416
Email: fong@zoology.ubc.ca
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