Hi all
I am staining mouse brain sections for PV and NeuN. My protocol has me perfusing the animals with 4% PFA, leaving the brains overnight in 4% PFA, then switching the brain to sucrose.
Yesterday, I perfused two animals and both perfusions went very poorly. The animals organs did not "inflate" and turn a pale color like they usually do, and when I extracted the brain it was pink and mushy.
Are these brains salvageable for IHC? I plan on leaving them in 4% PFA for 35-40 hours before switching them to sucrose, in the hopes of some fixation.
What do you all think?
Thanks
Hi Michael,
if you are fast enough to praparate the brains out of the scull and put them for 3 -5 days in 4% PFA and later for kryoprotection in 30% sucrose for another 3-5 days (the tissue has to be sunken on the ground of the vial), then it shoul work. If the mice are very small you have to think weather an immersion fixation in PFA would be better then a perfusion. Cresyviolett stained sections (Nissl's stain) can give you an impression of the quality of your tissue. If you see no so called dark neurons you can continue with your NeuN and PV IHC. But if you see alot af dark neurons the you would get in trouble with the PV IHC, because PV couldn't stain dark neurons. Good luck!
Hi Michael,
I inject PHA using a syringe calmly.
Alternatively, we think that PHA can supplement a mouse brain section by sinking it.
I would assume that "mushy" could mean necrosis/tissue compromise. I would not expect results to be reliable. If you proceed and use the tissue experimentally, be sure you note the aberration as it introduces an uncontrolled variable. I would omit it, most likely.
Similar situation happened to me once and I left the brain in 4% PFA for 3-5 days followed by 30% sucrose overnight. I stained Iba1 (fluorescent) and the result was fine. I think post-fixation is generally fine for IHC (both cryo and paraffin-embeded sections). Just remember to chop the tissue into smaller pieces so that the fixative can penetrate better. The concern is that residual blood in vessels trend to autofluoresce. But if you have a good, validated antibody which gives you enough signal-to-noise ratio, I think there will be no problem. Now since you mentioned the tissue was "mushy", it is up to you to decide whether the structural integrity of your regions of interest is still preserved.
Hi Michael,
We agree to the opinion of the DR. Sebastian Schmitt.
We can inject buffered formalin with a thin needle slowly.
Also, we can soak it in buffered formalin of enough quantity.
I commonly use buffered formalin.
Dear Michael,
I agree with Sebastian and Kathrina--get new mice. Unless, of course, this is somehow extremely valuable and rare material. Then you could try Ute's suggestion to screen them with Nissl staining first. Even then, you might go through all the work of sectioning and Nissl staining, yet still not be able to use them. Your time is also valuable, as well as the IHC reagents.
It might be a better use of your time to figure out why the perfusions went poorly, and work on a more reliable technique. Or, as suggested by some folks above, figure out a way to save a poor perfusion. I usually use the transcardial technique in mice (insert needle into L ventricle), and make sure that the wash step is going well before I switch to fixative. However, I always have a long cannula ready, in case it is not going well and I need to go thru the heart and into the aorta.
Good Luck,
Jill
Hi Michael
Try to inject heparin (intraperitoneal) before perfusion and you will never have this problem. I also prepare PBS+heparin for perfusion after intraperitoneal injection. The rate of formaldeid penetration is 1mm/8 hours. Immersion is not a good way for large specimens as the brain. Immersion is only for 2mm thick specimens.
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