BrdU preparation and administration

·       Prepare stock solution of BrdU 10mg/ml in sterile PBS.  BrdU powder is in the small -20°C fridge. Store the aliquots at -20°C in darkness for a maximum time of 3-4 months. Once you thaw the aliquoted vial keep it at 4°C for 5 days max.

·       Inject intraperitoneally 150-200 ul per mouse of BrdU 10mg/ml. Ideally, you want to inject 50ug/bodyweight.

·       Wait 2 hours and then sac the animal

 BrdU staining on cryosections

·       1/2 head fixed in 4% PFA 15 min at 4C

·       Wash 3X15' in PBS

·       Incubate 1/2 heads in 30% sucrose in PBS O/N at 4C

·       OCT embedding

·       Fix slides  in 4% PFA for 10 min at RT

·       Wash in 1X PBS 2x10’ at RT shaking. In the meantime warm up 2N HCl at 37°C

·       Place slides  in 2N HCl and incubate for 25’ at 37°C

·       Wash in 1X PBS for 5’ shaking

·       Blocking step: PBS + 0.1%Triton+ 2% NDS+ 1% BSA

·       Abs:  rat anti-BrdU (Abcam ab6326) diluted between 1:100 and 1:400 in PBS +0.1% triton +1% BSA, O/N

Day after:

·       Wash 0.1%Triton +1% BSA   2x10’

·       Abs: donkey anti rat-FITC    1:200   for 1 h at RT

·       Wash 0.1%Triton +1% BSA   2x10’

·       Vectashield +Dapi (do not use Hoechst 33342)