[Histonet] My perfusion problem

[Ignacio Ruz Caracuel]

Hi!

When I used to do whole body perfusion, it tooks me around 10 minutes to
perfuse with 0,9% salin and another 10 minutes with PFA. (Sorry, I haven´t
with me the rpm) but it was around 300 ml of each.

I think you have to increase the amount of 0,9% salin you perfuse (because
you have increased the circuit) to avoid clot formation.

I hope you find it useful and I wish you good luck!

Ignacio Ruz-Caracuel
Medical Student, Histology Intern Student
Faculty of Medicine,
University of Córdoba
SPAIN



2013/7/17 Leila Etemadi <leila.etemadi <@t> med.lu.se>

> Hi,
>
>  I am desperately looking for an answer to my problem in perfusion
> procedure.
>
> What is my problem?
>
> To explain that I thought first I may clear what is my usual routin.
>
> I used to perfuse rat's brain through this procedure:
>
> 1- Anesthetise animal with sufficient amount of pentobarbital.
>
> 2- Clamp the vessel lying beside vertebra in the back, and then using a
> perfusion pump with an 17 or 18 gauge blunted needle inserted into the
> ascending aorta.
>
> 3- Washout blood with 0.9% salin ( room temperature),  150-200 ml, 76 rpm
> speed ( usually I wait to see the out put solution from the heart is not
> looking so red/ blood look like).
>
> 4- Switch to the 4% cold paraformaldehyde ( with buffer, 7.4 pH) ( on ice
> cold), 150-200 ml, 46 rpm speed. ( some times I can see the formalin dance
> but not always !).
>
> 5- after using this amount of PFA normally the neck part is quite stiff,
> then animal will be decapitated. I extract the brain right away, postfix in
> PFA over the night ( 4°c in the refrigerator).
>
> 6- Move to the sucrose 20%. When I saw brain is sinking in the solution (
> which normally it happens in one and half day), I freeze the brain on dry
> ice quickly.
>
> The results of this procedure is good enough histology after dissection
> with cryostat ( 12-20µm).
>
>
>
> For my recent project I need brain and spinal cord both so basically I
> skip clamping the back
>
> along vertebra to circulate solution through whole body ( I didn't change
> any other steps, so I do
>
> exactly whatever I mentioned above except for spinal cord post fixation in
> PFA is about 2 hours).
>
> After sectioning ( 16µm), I've got a proper tissue from spinal cord but
> when it comes to the
>
> brain result is quite different !!, when I replaced my sections on the
> slide ( with plus charge),
>
> suddenly a lot of hole began to shaped in the tissue which practically
> ruined the entire
>
> piece..!!!,
>
>  First I had no clue for what I could see but then I noticed during post
> fixation procedure, when I
>
> transfer brain from PFA solution to the sucrose, brain is sinking right
> away ( it is not
>
> floating at all, as it suppose to do..) !!!!!
>
> So I've run another perfusion procedure but this time when I reached to
> the washing out by cold
>
> PFA I didn't decreased the pump speed ( as normally I decrease it from 76
> rpm to 46 rpm,
>
> this time I run whole procedure with the speed of 76 rpm), on the other
> hand I used more PFA
>
> solution ( about 350 ml PFA). After extracting brain I noticed brain was
> looking dried up.
>
> during post fixation and cryoprotection procedure (sucrose solution),
> sinking was looking normal,
>
> but after sectioning of course brain tissue was destroyed again!!!
>
> Now, I need your expert advices for my problem. I apologise for my naivety
> on this subject in advance.
>
>
> Humbly appreciate for your great time and attention in advance. I severely
> looking forward to your help and valuable tips.
>
>
> With the best,
>
> Leila
>
>
>
>
>
>
>
>
>
> Leila Etemadi
> M.Sc., Ph.D Candidate
> Neuronano Research Center (NRC)
> Lund University, BMC F10
> Sweden
>
> Tel: +46-46-2221503<tel:+46-46-2221503>
> E-mail: Leila.Etemadi <@t> med.lu.se<mailto:Leila.Etemadi <@t> med.lu.se>
>
>
>
>
>
>
>
>
>
>
>
> Sent from my iPad
>
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