Morrison lab, revised 3/31/04 Page 1
Protocol for BrdU labeling of adherent CNS or PNS stem cell cultures
References: Raff, M.C. et al Nature 333:562 (1988) and Molofsky, A.V. et al Nature 425,
To quantify proliferation (the percentage of cells that have replicated their DNA) in
adherent colonies, cultures can be pulse labeled with the thymidine analogue
Bromodeoxyuridine (BrdU), then fixed and stained to visualize BrdU labeled nuclei. This
can be followed with stains for other (compatible) markers.
All washes where PBS is indicated should be performed with PBS containing 0.01%
Igepal. The igepal reduces surface tension and prevents the cells from being ripped off
1. Pulse label with BrdU by adding 15 ul of a 1 mM BrdU stock per 1.5 ml of culture
medium (10uM final concentration). Return to the incubator for 1 hour.
2. Dump off medium and fix the plate at –20°C in 70% EtOH (in water) for 30
3. Wash three times in PBS containing 0.01% Igepal.
4. Denature the DNA by treating for 10 min in 2N HCl (made up in PBS.)
5. Wash 3 times in PBS containing 0.01% Igepal.
6. Neutralize acid by treating for 10 min in 0.1 M Sodium Borate (dissolved in 0.1 M
phosphate buffer and pH’d to 8.5).
7. Wash twice in PBS containing 0.01% Igepal, and once in PGN (see below).
8. Preblock at least 20 min in PGN (see recipe below).
9. Add primary antibody: anti:BrdU 1:100 (Caltag MD-5000). Rock for 30 min at RT.
10. Wash twice in PBS containing 0.01% Igepal, once in PGN.
11. Add secondary: goat anti mouse IgG 1:200 (Jackson 115-035-166). Rock for 30
min at RT.
12. Wash 3 times with plenty of acetate-imidazole buffer (recipe below).
13. Add Nickel-DAB solution to develop (about 0.7 ml per well of a six-well plate).
Wear gloves and avoid contact with NiDAB, it is toxic and carcinogenic. Monitor
over about 10 min as a colored precipitate forms – you should see BrdU labeled
nuclei begin to appear as the solution develops – they will be light purple at first,
and black later on.
14. Remove NiDAB solution to liquid waste, and rinse twice in PBS, once in PGN.
Note that the NiDAB solution is toxic and carcinogenic and therefore should be
disposed of as a hazardous waste, consistent with OSHA policy.
15. Counterstain with other antibodies as desired. In the case of CNS stem cell
colonies, continue at this point with the triple label protocol to label for neurons
16. Counterstain with DAPI (Sigma). DAPI stock solution is made to 1 mg/ml in water
and kept at -20˚C. Prepare DAPI 1/1000 in PGN and add to plates. No rinsing
Morrison lab, revised 3/31/04 Page 2
required. Note that when analyzing plates, BrdU labeled nuclei will usually have
little or no DAPI staining because the NiDAB deposition product blocks DAPI
To 500 ml of PBS add:
20 ml goat serum (heat inactivated, Gibco)
2 g BSA (FisherBiotech CAS 9048-46-8)
5 ml 10 % Igepal (NP-40; Sigma)
2.5 ml 10% NaN3 (sodium azide; sigma) Store at 4°C
Acetate Imidazole buffer:
87.5 ml Sodium Acetate 1M (pH 7.2)
25 ml imidazole 0.2 M, pH 9.2
450 ul Glacial Acetic Acid
add water to 500 ml
the pH should be 7.2, store the solution at 4°C
Nickel-DAB developing solution (10 ml):
This solution should be made immediately before use. Note: DAB is carcinogenic, be
especially careful when making DAB stock solution. All nickel DAB waste should be
disposed of as hazardous. Any spills should be wiped down with 100% bleach.
7 ml water
1.25 ml Sodium Acetate 1M
0.5 ml Imidazole, ph 9.2
0.3 ml diaminobenzidine (DAB) stock (10 mg/ml in water, stored in 1 ml aliquots at -20°C;
store the DAB powdered stock bottle in a dessicator in the dark as it is light sensitive)
30 ul freshly made 1% H2O2 solution (290 ul water + 10 ul 30% H2O2 stock)
Stocks and antibodies:
Mouse anti:BrdU: Caltag MD-5000
Goat anti-mouse IgG: Jackson immunoresearch115-035-166