Hi Carolina,

The best GFAP antibody that worked fo us (in mouse) was the one from abcam (rabbit polyclonal, ab7260).

I am not sure what is your exact protocol for the staining, but as organotypic sections are very thick, I would advice:

1) make sure that you include an extensive permabilization step (at least 3 days)

2) prolong the time of the primary and secondary antibody incubations

3) make sure that both primary and secondary incubations are done on a shaker

Glial scarring itself might indeed impact the penetration. As far as I am aware there are some methods that could inhibit glial scarring, but these are all treatmets that introduce additional variables to your experiments.

Good luck!

Hi Carolina, I work on organotypic cultures.

For the organotypic cultures is really important to wask them properly. Usally the day before the staining I wash them with PBS for the whole day, changing the pBS every 30 min.

The working protocol for me is to use a blocking-permeabilization solution. I do 2/4 hours of blocking and then the ABI O.N. The ABI is solubilized in the same solution of blocking (PBS, BSA 10%, NGS 0.5% (up to 1% depending on the AB), triton 0.3%). I would not increase the % of the triton on the culture because it will dissolve them.

Anyway if this does not help you in general for thiker brain sections (150/400 um) I use DMSO 0.5% to 1% and triton 1% this helps the penetration of the antibody.

The most important thing is that since the cultures are on peaces of memebrane, you have to make sure that you are putting the antibody on the slice and that your slice is not faced down but up.

You could also check the protocol on this paper: doi:10.1038/nprot.2006.180

Staining protocol for organotypic hippocampal slice cultures

Good luck