Immunofluoresent staining of cells in culture
Important notes:
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Use sterile cover glasses only.
All washes and incubations take place in a 24-well plate.
All washes are performed in a 500μl volume.
Blocking is performed in a 200μl volume.
Antibody incubations are performed in a 160μl volume.
Staining:
1. Fix cells in 1:1 Acetone;Methanol for 10min at R.T with gentle shaking.
2. Wash in PBS for 30-60sec.
3. Wash in 0.025% Triton X-100 in PBS 3x 5min with gentle shaking
(250μl Triton into 1L PBSx1).
4. Block in 3% normal goat serum in PBS for 1h at R.T (300μl normal goat
serum into 10ml PBSx1).
5. Discard blocking solution.
6. Add primary antibody diluted in 3% normal goat serum in PBS.
7. Incubate 1h at R.T or O.N at 4C.
8. Wash in 0.025% Triton X-100 in PBS 3x 5min with gentle shaking
9. Add secondary antibody diluted in 3% normal goat serum in PBS.
10. Incubate 1h at R.T.
11. Wash in 0.025% Triton X-100 in PBS 3x 5min with gentle shaking.
12. Dry cover glasses as much as possible.
13. Add 10μl ready to use DAPI + mounting to a slide, put the cover glass on
the slide (cells facing downwards to the slide).
14. Slides can be kept at 4C for a few months.