Brdu免疫组织化学染色分析BrdUincorporationassay

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