What if grows cells in brdu solution for more than 24hrs?
Answers
Dechorionate and chill 15 m on ice in E3.
2.
Prepare cold 10 mM BrdU⧸15% Dimethylsulfoxide (DMSO) in E3 and chill on ice. Place embryos in BrdU solution and incubate 20 m on ice to allow uptake of BrdU.
3.
Change into warm E3 and incubate exactly 5 m, 28.5 °C. Note: Longer incubation times will result in more cells being labeled.
4.
Fix 2 h at room temperature in PFA. Longer fixation may decrease the staining.
5.
Transfer to methanol at −20 °C overnight. All subsequent steps are performed at room temperature unless otherwise noted.
6.
Rehydrate in graded methanol:PBST series (3:1, 1:1, 1:3) for 5 m each.
7.
Wash 2× in PBST, 5 m.
8.
Digest embryos in 10 ug⧸ml Proteinase K, 10 m.
9.
Wash PBST. Refix in PFA for not more than 20 m.
10.
Wash quickly 3× in H2O, then 2× in 2N HCl.
11.
Incubate 1 h in 2N HCl. This step denatures the labeled DNA to expose the BrdU epitope.
12.
Rinse several times in PBST. It is important to bring the pH back up to approximately 7 before adding blocking solution.
13.
Block for 30 m in BrdU blocking solution.
14.
Incubate in monoclonal anti-BrdU antibody at a dilution of 1:100 in BrdU block for 2 h at room temperature or overnight at 4 °C. (If carrying out simultaneous BrdU⧸pH3 staining, add the primary anti-pH3 antibody as described in Section B, except that BrdU block is used.)
15.
Wash 5 × 10 m in PBST.
16.
Incubate 2 h at room temperature with horseradish peroxidase or fluorophore-conjugated anti-mouse secondary antibody. (For simultaneous BrdU⧸pH3 stain, add a fluorescent anti-rabbit antibody as well.)
17.
Wash 5 × 10 m in PBST. Develop with diaminobenzidine if using HRP-conjugated secondary antibody.