Immunofluorescence assay (IFA)

Seed the HeLa cells onto HistoGrip (ThermoFisher) coated coverslips in growth media in 6 well plates (DMEM supplemented with 10 % (v/v) FBS, 1 % Penicillin-Streptomycin, 25millimolar (mM) HEPES, GlutaMAX (Life Technologies) and non-essential amino acids (Life Technologies)).

Aspirate off the culture media and add 700µL of fixation buffer to each well and incubate on a side-to-side shaker for 0h 10m 0s.

Aspirate off the fixation solution and wash three times with 1x PBS.

Permeabilise the cells with Permeabilization buffer (2mL for each well) for 0h 10m 0s on a side-to-side shaker.

Aspirate off the Permeabilization buffer and wash once with 1x PBS.

Aspirate off PBS and incubate the cells with Blocking buffer (700µL for each well) for 0h 15m 0s on a side-to-side shaker.

Aspirate off the Blocking buffer, wash once with 1x PBS and once with permeabilization buffer.

Aspirate off the permeabilization completely and put the 6 well plates on a flat bench.

Add the primary antibodies (diluted to the right concentrations in Blocking buffer; 15µL/well) onto the glass coverslips and incubate for 1h 0m 0s - 2h 0m 0s depending on the strength of the antibodies.

Wash the coverslips twice with 1x PBS and once with permeabilization buffer.

Aspirate off the permeabilization completely and put the 6 well plates on a flat bench.

Add the relevant secondary antibodies (diluted in Blocking buffer to 1/200-1/500; 15µL/well) onto the glass coverslips and incubate in the dark for 1h 0m 0s – 2h 0m 0s.

Wash three times with 1x PBS and mount the coverslips on microscope slides.

Use a yellow tip, drop one small drop off mounting medium onto a microscope slide.

Pick out one coverslip from a well (still in 1x PBS) and place it onto a piece of whatman paper so that the side that the cells grew on is facing up.

Take the microscope slide with a drop of mounting medium and put it on the coverslip and gently push it down

to remove excessive mounting medium.

Seal the edges with nail polish, let it dry and store in a microscope box at 4°C prior to imaging.