Question (71958) | Anti-Geminin antibody (ab104306)

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The following protocol was used with ab104306 for ICC/IF:

Preparation of slides:

1. Grow cultured cells on sterile glass cover slips at 37°C overnight.

2. Wash sample with PBS twice.

3. Fix cells for 15 minutes with 2 mL of 4% paraformaldehyde solution (pH 7.4 with NaOH in PBS).

Step-by-step procedure:

1. Permeabilize cells by incubating for 15 minutes on ice with 2 mL of 0.1% Triton X-100 in PBS.

2. Wash cells 3 times with PBS.

3. Incubate cells for 1 hour with normal goat blocking serum (1:20 in PBS).

4. Introduce primary antibodies (in appropriate dilutions) to the sample.

5. Incubate for 4 hours at room temperature or at 4°C overnight.

6. Wash with PBS for 5 minutes. Repeat 5 times.

7. Incubate cover slips in fluorescein-conjugated secondary antibodies in 5% normal blocking serum in PBS in a dark humidity chamber at 4°C for 1 hour.

(Perform all subsequent washes under dim and ambient light source.)

8. Wash sample thoroughly with PBS. Each wash lasting 5 minutes. Repeat 6 times.

9. Counter stain sample with DAPI at room temperature for 30 minutes.

10. Wash sample with PBS. Each wash lasting for 2 minutes. Repeat 3 times.

11. Mount sample by inverting them onto mounting medium on glass slides.

12. Store slides in dark at 4°C.


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