Brdu免疫组织化学染色分析 BrdU incorporation assay

Enzyme Immunostaining for BrdU:

1. Wash frozen sections with PBS 2x.
2. Put them in 0.1% Pepsin in 0.1N HCL (in PBS) at 37�C for 50min.
3. Then in 0.3% hydrogen peroxide in PBS, 30min. Then rinse with PBS 3x.
4. Then in 5% normal goat serum in PBS with 1% BSA at RT for 1 hour.
5. Mouse anti-BrdU diluted to 1:1000 with 1% NGS and1%BSA in PBS, incubateat 4�C overnight.
6. Rinse with PBS 3x.
7. Goat Biotinylated anti-Mouse IgG 1:200 in 1% NGS, 1%BSA in PBS, incubate at RT for 1 hour. Rinse in PBS 3x.
8. 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).
9. Rinse with PBS 3x.
10. 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.
11. Rinse with distilled water 3x. Counterstain and mount.

Culture Cell Fluorescence Immunostaining for BrdU:

1. Wash plates with PBS 3x.
2. Fix them in 4%PFA, 20 min at RT.
3. Wash them in PBS 3x.
4. 0.5% Triton X-100 in PBS, 15min at 4�C
5. Wash slides in PBS 3x.
6. 2N HCL( in Distilled water) 30min at 37�C( DNA denaturation).
7. Wash in PBS 5x
8. Incubate slides with 1%BSA in PBS at RT for 30min.
9. Mouse anti-BrdU diluted to 1:1000 with 1% NDS and1%BSA in PBS, incubate at RT for 2hrs.
10. Wash with PBS 3x.
11. 1: 100 dilution of Rhodamine(Tritc) conjugated Donkey anti mouse IgG with 1% NDS,1%BSA in PBS, incubate at RT for 1 hr.
12. Wash in PBS 3x
13. Mount with 50% glycerol in PBS.

Culture Cell Enzyme Immunostaining for BrdU:

1. Wash plates with in PBS 3x.
2. Put 4%PFA 20 min at RT.
3. Wash with PBS 3x.
4. 0.5% Triton X-100 in PBS, 15min at 4�C
5. Wash with PBS 3x.
6. Incubate with 2N HCL( in Distilled water) 30min at 37�C( DNA
   denaturation).
7. Wash with PBS 5x
8. 1% BSA ,incubate for 30min at RT.
9. Mouse anti-BrdU diluted to 1:1000 with 1% NGS,1%BSA in PBS,
   incubate at RT for 2hrs.
10. Wash withPBS 3x.
11. Dilute Goat Bionytilated anti-Mouse IgG to 1:200 with1% NGS and
    0.1% BSA in PBS, incubate at RT for 1 hour. Rinse with PBS 3x.
12. ABC at RT for 1 hour. Rinse in PBS 3x.
    ( ABC solution: 5ml PBS with 1 drop A and 1 drop B, mix well)
13. 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.
14. Rinse with distilled water 3x.
15. Countstain with Mayer’s Hematoxylin