For an outstanding, detailed BrdU protocol see Rothaeusler and Baumgarth (2006)
Cytometry 69A:249
This article is great. It compares four staining protocols that rely on purchased kits ($$) to homemade reagents. Not only do the authors examine efficacy of BrdU detection but also the effects of different fixation and permeabilization methods on quenching of surface fluorochromes.
The bottom line: pay the extra money to use the BrdU kit from PharMingen (#559619
FITC, also available as APC).
Also CRITICAL is DNAse concentration. Use DNAse I Worthington: catalog # LS002139
(100 mg). It comes as a lyophilized powder, and I prepare it in PBS containing Ca and
Mg. Prepare the DNAse fresh right before use.
Special notes:
1) We have gotten good results with the BrdU kit with the exception of DNAse problems.
Not only do we run out of this reagent first but it seems to give variable quality results.
Since switching to Worthington DNAase, prepared freshly as above, our results are great. So now we use the BrdU kit with Worthington DNAse.
2) As an easy positive control for effective BrdU resolution, inject mice with BrdU (200 ul of a 3 mg/ml solution) 1 hour prior to sacrifice. Stain with CD4 and CD8, ~8% of double positive thymocytes will be BrdU+ and these will be localize to the population with higher FSC.
3) If you run out of BrdU in the PharMingen kit, use Sigma Aldrich #858811