Immunofluorescence staining

Remove medium.

Wash with 1× PBS.

Remove PBS and add 4% PFA to the cells.

Incubate 0h 10m 0s at Room temperature.

Collect PFA.

Wash 1× PBS.

Wash with 1× PBS for 0h 5m 0s at Room temperature. (1/3)

Wash with 1× PBS for 0h 5m 0s at Room temperature. (2/3)

Wash with 1× PBS for 0h 5m 0s at Room temperature. (3/3)

Add the same volume of 8% PFA as medium is in the well to the well.

Incubate 0h 10m 0s at Room temperature.

Collect PFA in a falcon.

Wash in 1× PBS.

Wash with 1× PBS for 0h 5m 0s at Room temperature. (1/2)

Wash with 1× PBS for 0h 5m 0s at Room temperature. (2/2)

Remove 1X PBS.

Add 300µL/well of blocking solution.

Incubate at least 1h 0m 0s at Room temperature.

Prepare antibody in blocking solution containing 5% NGS.

Put a drop (50µL) of Primary antibody solution on the parafilm surface.

Remove the coverslips from the plate and gently put it upside-down on the antibody drop.

Incubate 1h 0m 0s at 4°C.

Wash:

Wash for 0h 5m 0s in PBS + Triton X-100 0.1%. (1/3)

Wash for 0h 5m 0s in PBS + Triton X-100 0.1%. (2/3)

Wash for 0h 5m 0s in PBS + Triton X-100 0.1%. (3/3)

Prepare secondary antibody in blocking solution containing 5% NGS.

Put a drop (50µL) of secondary antibody solution on the parafilm.

Take the coverslips and put it upside-down on the antibody drop.

Incubate 1h 0m 0s at Room temperature in the dark.

Transfer the coverslip to a 24-well containing 500µL 1X PBS + Triton X-100 0,1%.

Incubate for 0h 5m 0s at Room temperature (in the dark).

Dilute DAPI 1:10000 in 1X PBS.

Remove PBS and incubate with DAPI for 0h 5m 0s at Room temperature in the dark.

Wash.

Wash with 1X PBS. (1/2)

Wash with 1X PBS. (2/2)

Mount the slides:

Put a drop (10µL) of DAKO mounting reagent on the slide.

Take out the coverslip from the plate.

Dry it by gently tapping the coverslip’s edge on a lens-cleaner tissue.

Gently put the coverslips upside-down on the DAKO drop.

Leave it dry (24h 0m 0s, DARK, Room temperature).

Store in a slide-box at 4°C.