brdU double labelling - (Dec/29/2004 )

I would be grateful if someone could help improve my staining. I am interested in BrdU-NeuN double labelling on mouse brain. My BrdU protocol is 50mg/kg (sigma) twice a day for 2 days intraperitoneally, the brain is sliced at 40 microns and stained free floating. Denaturation is with 2M HCl for 30 mins with a 30 min wash in PBS (3x10 mins). Sections are blocked in 5% normal donkey serum and MOM (mouse on mouse) kit (Vector) in TxTBS for 2 hours. The primary antiBrdU is an abcam antibody (polyclonal raised in sheep) 1/1000 (I have also tried fitzgerald) which is incubated together with anti NeuN at 1/400 (chemicon, raised in mouse) overnight at 4 degrees (with 1% NDS and MOM kit in TxTBS). Each wash is an hour long. The secondaries are applied seperately (alexo fluor donkey anti sheep 488 and goat anti mouse 568). The staining i get is just perceivable and there is a lot of background (but much better using DAB). A control whereby I swapped the secondaries showed minimal but detectable cross reaction. Amplification didnt help much.
I would very much appreciate any comments
Best wishes
wendy

Wendy,

I haven't performed double labeling with BrdU and Neu-N but have used BrdU antibodies and some against GFAP and beta-tubulin. I always perform one entire labeling reaction before starting the other (i.e., GFAP primary and secondary then anti-BrdU). This way, you could take a quick look at the first reaction to ensure that you are getting the signal you want. It could be that your denaturation protocol is having a negative impact on the Neu-N staining. I would try the Neu-N first, all the way through, then the BrdU. Reverse the sequence on another section and see if there is any difference. Hope this helps.

Chris