Brdu免疫组织化学染色分析 BrdU incorporation assay
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Enzyme Immunostaining for BrdU:
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- Wash frozen sections with PBS 2x.
- Put them in 0.1% Pepsin in 0.1N HCL (in PBS) at 37C 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 4C 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:
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- 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 4C
- Wash slides in PBS 3x.
- 2N HCL( in Distilled water) 30min at 37C( 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:
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- 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 4C
- Wash with PBS 3x.
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Incubate with 2N HCL( in Distilled water) 30min at 37C( DNA
denaturation). - Wash with PBS 5x
- 1% BSA ,incubate for 30min at RT.
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Mouse anti-BrdU diluted to 1:1000 with 1% NGS,1%BSA in PBS,
incubate at RT for 2hrs. - Wash withPBS 3x.
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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. -
ABC at RT for 1 hour. Rinse in PBS 3x.
( ABC solution: 5ml PBS with 1 drop A and 1 drop B, mix well) -
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.
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Countstain with Mayer’s Hematoxyli
