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

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:

1. 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