[Histonet] My perfusion problem

[Leila Etemadi]

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>











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