Dear Jason Patel,

your proposal is feasible and performed here in our Institute.

Persons are doing this, when they have only small amounts of animals, for example because only a small proportion of the litter is transgenic.

However, if you can obtain enough mice for your experiments I would recommend transcardial perfusion with the fixative, postfixation and then followed by cryosectioning or paraffine embedding. For IHC you will only get the best results then.

Sincerely Yours

Stefan

Stefan, 

Thank you kindly for your reply. 

To clarify, I do have a small litter to work with so efficiency is essential. In your lab, how do you accomplish both techniques?

Do you cut in half, flash freeze one side and then just immerse the other half in 4‰ pfa? is saline perfusion utilized prior to the dissection to clear the blood? How long do you let it sit in pfa? How long do you place that fixed tissue in 30% sucrose?

Hello,

I personally don't do it actually.

But I know the procedure:

Dissect brain without any perfusion. Cut sagitally (perhaps by using a brain matrix, but they are expensive), one half is directly frozen or put in RNA-later for mRNA studies and the other half is put directly into 3.7% PFA (solved in PBS 0.1M, pH 7.4) for about 24 h (the next day) for fixation. Then I put them in 30% sucrose (solved in PBS 0.1M, pH 7.4) over night at 8°C until they sank to the bottom of the glass. Then I snap-freeze them in isopentane at  -50 to minus 80°C.

Ready for cryostat-sectioning. I then perform IHC with free-floating sections.

The disadvantage is the blood in the brain vessels. Therefore I perfuse for IHC!!!

I'm anatomist and prefer a good morphology in comparison to nice bands on a western.

;-)

Sincerely yours

Stefan

Thank you again Stefan :)