I have tissue from mice that were injected with EDU and then BRDU, sectioned at 40um.  I am using the Click-iT EDU kit and trying to double label with BRDU (and ideally triple label for Parvalbumin and other neuronal markers in the future).  I've tried two methods of labeling with sections free floating:

Any advice?  Thanks!

Details on methods

Method 1:

Wash 2X PBS

Permeablize 3XPBS-Triton

EDU Click-iT reaction for 30 min (in dark)

DNA denature: HCL2N for 30 min

Wash 2XPBS

Wash1X PBS-Triton

Block: PBS-Triton + Donkey Serum for 1 hour

Primary anti-BRDU overnight in block

Wash 3XPBS-Triton

Incubate  secondary in PBS-Triton for 90min (in dark)

Wash 3xPBS

Mount

Method 2:

Wash 2X PBS

Wash 1X PBS-Triton

DNA denature: HCL2N for 30 min

Wash 2XPBS

Wash 1X PBS-Triton

Block: PBS-Triton + Donkey Serum for 1 hour

Primary antoi-BRDU overnight in block

Wash 3XPBS-Triton

Incubate  secondary in PBS-Triton for 90min (in dark)

EDU Click-iT reaction for 30 min (in dark)

Mount