Assay Procedure
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- 1. Fixation: 4?% paraformaldehyde (PFA) or 1:10 formalin (37?% formaldehyde, 10-15?% MetOH) in PBS, 10?min., RT.
- 2. Wash 3x with PBS containing 0.1?% Tween 20 (PBST). Critical: do not let coverslips "dry".
- 3. Permeabilisation: PBS containing 0.5?% Triton X-100, 5?min., RT. Alternatively permeabilise by incubating in 100?% methanol for 5?min at -20?°C.
- 4. Wash 2x with PBST.
- 5. Blocking: 4?% BSA in PBST, 10?min, RT.
- 6. GFP-Booster incubation: dilute GFP-Booster 1:200 in blocking buffer and incubate 1 h, RT. Note: For multiplexing protocols you can combine GFP-Booster with any other antibody.
- 7. Wash 3x 5-10?min in PBST.
- 8. If required counterstain with DNA fluorescent dyes, e.g. DAPI.
- 9. Before mounting coverslips can be very briefly rinsed in water to prevent salt crystals to form.
- 10. Mount in VectaShield (Vector Labs) or other mounting media with anti-fading agents and seal mounted coverslips with clear nail polish. Please note: Optimal dilutions/ concentrations should be determined by the end user
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