Indirect immunofluorescence staining of adherent cells
Except where noted all steps are performed at room temperature.
Use Delbecco PBS (with magnesium/calcium)
Use 5 ml of buffer per wash. Make sure the coverslip is completely submerged at all steps.
- Transfer coverslips (cell-side up) to individual small weigh boats containing PBS.
Wash twice for 1 min. each.
- Fix cells in 3.7% formaldehyde in PBS, 10 min.
2 ml 37% formaldehyde
18 ml PBS
- Wash cells in PBS, 1 min. Repeat.
- Permeabilize cells in 5 ml of 0.2% Triton X-100, 5 min. Slow shake.
400 ml 10 % TX100
19.4 ml PBS
- Wash cells in PBS, 5 min. Repeat.
- Pre-block coverslips in 1 ml PBS with 1% serum. RT, 20 min.
10 ml goat serum (GIBCO)
1 ml PBS
Turn coverslips cell-side down during incubation to avoid drying.
- Incubate in primary antibody in 1 % goat serum in PBS. 37°C, 1 hr.
e.g. 2 ml anti-LIMA antibody (=1:500 dilution)
10 ml goat serum
1 ml PBS
Incubate the coverslips cell-side down in a moist chamber at 37°C.
- Turn the coverslips back over. Wash in PBS, 5 min. Repeat.
- Incubate with secondary antibody in 1 % goat serum in PBS. 37°C, 45 min.
5 ml goat anti-rabbit IgG-FITC antibody
5 ml goat serum
500 ml PBS
Incubate coverslips cell-side down in a moist chamber.
- Turn coverslips over (cell-side up).
Wash three times with PBS, 5 min.
- Fix in 3.75% formaldehyde in PBS. RT, 30 min.
- Quench in 5 ml 50 mM NH4Cl. RT, 15 min.
1.25 ml 1 M NH4Cl
23.75 ml PBS
- Wash in PBS. 3 min. Repeat 5 x.
- Mount in a drop of Vectashield (Vector Laboratory CA) (cell-side down). Seal edges with nail polish.
Store slides in the dark at 4°C.