Immunofluorescence for adherent cells

Fix cells with 4% (v/v) PFA in PBS for 0h 10m 0s.

Note: Discard PFA in a special container and not in the drain.

Quench PFA by using a 50millimolar (mM) NH₄Cl solution in PBS for 0h 10m 0s.

Permeabilize cells with 0.25% (v/v) Triton X-100 in PBS for 0h 10m 0s.

Block with 4% (v/v) BSA in PBS for 1h 0m 0s.

Note: At this step the protocol can be paused. Blocking can also be done overnight.

Other blocking solutions can be used, such as goat or donkey serum matching with the species of the secondary antibodies used.

Incubate primary antibodies in 4% (v/v) BSA in PBS for 1h 0m 0s.

For adherent cells on glass coverslips, the antibody solution is drop seeded on parafilm and the coverslip is placed upside down on the antibody solution using the inverted tweezers. For the 22x22 mm coverslips, 100 ul of antibody solution is sufficient for the whole coverslip to be incubated with antibody. For cells growing on plastic the antibody solution can be added in the well/dish.

Wash cells with PBS for 0h 5m 0s three times.

Incubate secondary antibodies in 4% (v/v) BSA in PBS for 1h 0m 0s.

For immunofluorescence, we usually use Alexa dye secondary antibodies (Thermo Scientific) at a 1:500 dilution.

Wash cells with PBS for 0h 5m 0s three times.

Wash cells with ddH₂O to remove excess salt and protein crystals.

For cells on coverslips, the coverslips are dipped into a beaker with ddH₂O four times and excess ddH₂O is removed by touching the edge of the coverslips on absorbent paper.

Add the coverslip on a glass slide that has been previously drop seeded with Prolong Diamond mountant. Place coverslips inverted and avoid trapping any air in the process. Allow to air dry overnight in a dark environment.

For the 22x22 mm coverslips, 50 ul of mountant is sufficient. Pipette mountant with a cut tip to avoid bubbles.

Seal the coverslips the next day using clear nail polish.