In my experience,

- you shouldn't need any antigen retrival pre-treatment for any of the markers you want to stain. Especially on a P6 brain, these stainings should be piece of cake.

- your DCX seems to work. The high background might be due to two factors: the serum you use for blocking, and the fact that you add the fluorescent-conjugated secondary antibody before the acidic treatment.

so the protocol I suggest is

- make a good blocking with 10% serum, 0.4% Triton-X in phosphate buffer, 2 hours RT. for all the incubation steps, use 2% serum.

- primary O/N 4C anti-DCX, followed by 4 hrs 4C of the respective secondary antibody biotinylated.

- Acidic treatment followed by borate buffer wash, and 6x washes in PB

- primary anti NeuN and anti BrdU O/N 4C (they can be done together), followed by the respective fluorescent secondaries 4hrs 4C

- now, you develop the DCX staining by using a fluorescent-conjugated streptavidin, 30 minutes RT in PB-TritonX. it's very important not to use any serum in this step as it would deactivate the biotin-streptavidin binding.

- between each step listed above, extensive washes in PB.

- also, use a positive control for DCX, like some frontal sections where the rostral migratory stream should be visible.

Best,

Roberto

And by the way, 15 minutes HCl 2M 37C is more than enough to open up the DNA. longer incubation protocols will only deteriorate the tissue, no pre-treatment is necessary. the, 30 minutes wash in pH 8.5 borate buffer.

The high background may be caused by contaminated serum in your blocking buffer.  Through prolonged use you may have contaminated your stock serum.  Take the serum you use, and filter through (0.2um), then try again.  Good luck!

I think DCX signal in p6 mice in your experiment is correct. I did developmental expression of DCX in mice.