Ab-10 Rapid HiLyte Fluor 647 Labeling Kit

Fluorophore labeling

0-5°C

ambient temperature

FCM; Microscopy

Mitochondria Immunostaining
1. HeLa cells were seeded on a μ-slide 8 well (ibidi) and cultured overnight at 37 °C in a 5% CO2 incubator.
2. The cells were washed with PBS three times, and 4% paraformaldehyde in PBS was added to the μ-slide.
3. The μ-slide was incubated at room temperature for 15 minutes.
4. The cells were washed with PBS three times, and 1% Triton-X in PBS was added to the μ-slide.
5. The μ-slide was incubated at room temperature for 30 minutes.
6. Once the cells were washed with PBS three times, a blocking solution prepared with PBS was added to the μ-slide.
7. The cells were then incubated at room temperature for 1 hour.
8. Biotin conjugated anti-mitochondria antibody was diluted 50 times with the blocking solution. 　
※Anti-mitochondria antibody was purchased from Abcam (Product Code: ab3298) .
9. The supernatant was discarded and the solution (step 8) was added to the μ-slide.
10. The μ-slide was incubated at 0-5°C overnight.
11. The supernatant was discarded and the cells were washed using PBS-T three times.
12. 0.2 µg/ml peroxidase conjugated streptavidin was added to the μ-slide.
13. The μ-slide was incubated at room temperature for 1 hour.
14. The supernatant was discarded and the cells were washed using PBS-T three times.
15. The cells were washed using Tris buffer (TB, 50 mmol/l, pH 7.5) three times.
16. The supernatant was discarded and DAB solution [0.2 mg/ml DAB (SKU:D006), 0.003% H2O2 , 50 mmol/l Tris (pH 7.5)] was added to the μ-slide.
17. The μ-slide was incubated at room temperature for 10 minutes.
18. After the cells were washed using TB three times, TB was added to the μ-slide.
19.The cells were observed under a microscope.