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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
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Three-μm thickness kidney cryosections were fixed for 15 min in 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 in 1× phosphate-buffered saline (PBS) for 5 min at room temperature. After blocking with 2% donkey serum for 60 min, the slides were immunostained with the primary antibodies. Cells cultured on coverslips were washed twice with cold 1× PBS and fixed with cold methanol/acetone (1:1) for 10 min at −20 °C. After three times of extensive washing with 1× PBS, the cells were treated with 0.1% Triton X-100 for 5 min, blocked with 2% normal donkey serum in 1× PBS buffer for 40 min at room temperature, and incubated with antibodies |
Cells were also stained with 4′,6-diamidino-2-phenylindole to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i epi-fluorescence microscope equipped with a digital camera or Zeiss LSM710 (Zeiss). |
| Protocol tips |
| Three-μm thickness kidney cryosections were fixed for 15 min in 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 in 1× phosphate-buffered saline (PBS) for 5 min at room temperature. After blocking with 2% donkey serum for 60 min, the slides were immunostained with the primary antibodies. Cells cultured on coverslips were washed twice with cold 1× PBS and fixed with cold methanol/acetone (1:1) for 10 min at −20 °C. After three times of extensive washing with 1× PBS, the cells were treated with 0.1% Triton X-100 for 5 min, blocked with 2% normal donkey serum in 1× PBS buffer for 40 min at room temperature, and incubated with antibodies |
| Downstream tips |
| Cells were also stained with 4′,6-diamidino-2-phenylindole to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i epi-fluorescence microscope equipped with a digital camera or Zeiss LSM710 (Zeiss). |
| Upstream tips |
Protocol tips |
Downstream tips |
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- Incubate membrane with 5% non-fat milk for 1 h at room temperature before adding primary antibody.
- Dilute Ab at 1:50 and incubate for overnight at 4C. |
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| Protocol tips |
- Incubate membrane with 5% non-fat milk for 1 h at room temperature before adding primary antibody.
- Dilute Ab at 1:50 and incubate for overnight at 4C. |
| Upstream tips |
Protocol tips |
Downstream tips |
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- Before adding primary antibody, incubate membrane with 5% non-fat milk for 1 h at room temperature.
- Incubate primary antibody overnight at 4°C |
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| Protocol tips |
- Before adding primary antibody, incubate membrane with 5% non-fat milk for 1 h at room temperature.
- Incubate primary antibody overnight at 4°C |
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