I am using the Abcam antibody ab4680 on mouse brain tissue fixed via PFA perfusion. The protocol we are using is the same as in http://dx.doi.org/10.1016/j.cell.2015.11.025. It's a staining method for multiple rounds of labeling of thick sections, and it's worked beautifully for lectin, GFAP, and NeuN.
Do you know of any reasons off the top of your head why this might not be working, or could anyone suggest alternate antibodies?
Thanks!
edit: currently the only NF staining is some random background stuff. othewrwise, nothing.
If not your antibody staining method maybe your paraffin sectioning method is avoiding the staining
Dear Aria,
did you include a permeabilization step (0.25% Triton X-100, 30min) step?
Perhaps also fixation in Bouin´s fluid could help.
Best regards, Daniela
Hi,
Has your antibody undergone any freeze thaw cycles?
I have never performed the protocol described, but have consistently had issues getting structural proteins (Tau, Map2, NF) to stain effectively in PFA/PE sections.
One NF antibody that has worked well for me in cell cultures and muscle sections is from Sigma. https://www.sigmaaldrich.com/catalog/product/mm/mab1621?lang=en®ion=US
I've attached two images (cell culture-- NF is shown in green and in muscle prep- NF is shown in cyan).
I use this antibody at a dilution of 1:250
In muscle sections, NF seems to work best with a shorter PFA fixation time.
Best of luck! I hope this helps.
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