[Histonet] rat perfusion

Wed Dec 19 15:24:31 CST 2007

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
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University of British Columbia
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Canada 

Ph:  (604) 822-5990
Fax: (604) 822-2416
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