Antibody-Based Technologies > Antibody B

BDNF IHC?

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hbrenhouse:
Hi there,

I would like to start an IHC stain for BDNF, but I've heard it is extra-tricky.  Does anyone have any experience with this in rat brain?

Thanks for any advice
Heather

Jossy:
Hi, I've realized a IHC for NGF, and works well, maybe my protocol works for you too. :D
(criostate slides of 40 micrometers)

Wash 15 min in PBS (0.1M, pH 7.4) at RT
Incubate 15 min in 0.3% H2O2 (in PBS) at RT
Wash 5 min in PBS at RT
Incubate 3 times in EtOH 50% (in PBS), 10 min each, at RT
Wash 15 min in PBS, at RT
Incubate 1 hr in PBS + 0.2% Triton x-100 / 5% Normal Serum at RT
Incubate with primary antibody (1:500), in PBS + 0.2% Triton x-100 / 2% Normal Serum at 4 C O.N.
Wash 3 times in PBS , 15 min each, at RT
Incubate with secondary biotinylated antibody (1:500), in PBS + 0.2% Triton x-100 / 0.1% NS, 2 hrs at RT
Wash 3 times in PBS , 15 min each, at RT
Incubate one and a half hour with ABC Kit (1:110) diluted in PBS, at RT (mix 30 min before use)
Wash 3 times in PBS , 15 min each, at RT
Incubate in 0.04% DAB + 0.01% H2O2 + NiCl2 0.06%, 10 min at RT (discard to chlorine solution)
Wash 3 times in PBS , 15 min each, at RT (discard to chlorine solution)

hbrenhouse:
Thanks, Jossy!  I've heard that there are few, if any, good BDNF antibodies out there, but if I find one I will give your protocol a try.  Thanks again

SGlerup:
We have used an antibody against the proform of BDNF (proBDNF) that works very well for IHC on mouse embryo and brain sections.
It is a rabbit polyclonal from Alomone.

I have reviewed it on www.pabmabs.com

A postdoc from our lab has also used BDNF mAb #9 from Yves Barde's lab (Matsumoto et al. 2008 Nat Neurosci) with success on primary hippocampal neurons treated with KCl.

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