Enzyme Immunostaining for BrdU:
Wash frozen sections with PBS 2x. Put them in 0.1% Pepsin in 0.1N HCL (in PBS) at 37�C for 50min. Then in 0.3% hydrogen peroxide in PBS, 30min. Then rinse with PBS 3x. Then in 5% normal goat serum in PBS with 1% BSA at RT for 1 hour. Mouse anti-BrdU diluted to 1:1000 with 1% NGS and1%BSA in PBS, incubateat 4�C overnight. Rinse with PBS 3x. Goat Biotinylated anti-Mouse IgG 1:200 in 1% NGS, 1%BSA in PBS, incubate at RT for 1 hour. Rinse in PBS 3x. Incubate with ABC reagent in PBS containing 0.1% Tween 20 at RT for 1 hour.. (ABC reagent solution: 5ml PBS/0.1% Tween 20 with 1 drop A and 1 drop B, mix well). Rinse with PBS 3x. DAB reaction: 2.5ml of distilled water + 1 drop Buffer + 2 drop DAB solution + 1 drop hydrogen peroxide. ~Needs about 8 min to stop DAB reaction. Rinse with distilled water 3x. Counterstain and mount.
Culture Cell Fluorescence Immunostaining for BrdU:
Wash plates with PBS 3x. Fix them in 4%PFA, 20 min at RT. Wash them in PBS 3x. 0.5% Triton X-100 in PBS, 15min at 4�C Wash slides in PBS 3x. 2N HCL( in Distilled water) 30min at 37�C( DNA denaturation). Wash in PBS 5x Incubate slides with 1%BSA in PBS at RT for 30min. Mouse anti-BrdU diluted to 1:1000 with 1% NDS and1%BSA in PBS, incubate at RT for 2hrs. Wash with PBS 3x. 1: 100 dilution of Rhodamine(Tritc) conjugated Donkey anti mouse IgG with 1% NDS,1%BSA in PBS, incubate at RT for 1 hr. Wash in PBS 3x Mount with 50% glycerol in PBS.
Culture Cell Enzyme Immunostaining for BrdU:Wash plates with in PBS 3x. Put 4%PFA 20 min at RT. Wash with PBS 3x. 0.5% Triton X-100 in PBS, 15min at 4�C Wash with PBS 3x. Incubate with 2N HCL( in Distilled water) 30min at 37�C( DNA