Immunofluorescence of macrophages and microglia

Plate 50,000 iPSC derived macrophages or microglia or Neurons or BMDM’s cells on 22 x 22 mm glass coverslips in 24-well dishes in respective culture media.

Incubate at 37°C in 5% CO₂.

Pre-warm a solution of 4% paraformaldehyde to Room temperature.

Aspirate media from cells and immediately proceed to step 5.

Add pre-warmed 4% paraformaldehyde to cells.

Incubate for 0h 15m 0s at Room temperature.

Remove 4% paraformaldehyde solution and dispose of it in an appropriate chemical waste container.

Rinse each well with 3X 1mL PBS. Remove rinse and dispose of it in an appropriate chemical waste container.

Add 0.1% (v/v) saponin + 5% (w/v) BSA in PBS and incubate for 1h 0m 0s at Room temperature to permeabilize cells.

Aspirate 0.1% (v/v) saponin + 5% (w/v) BSA in PBS solution.

Prepare respective antibody dilutions in 0.1% (v/v) saponin + 5% (w/v) BSA in PBS solution. mouse LAMP1 (DSHB #ID4B) 1:200 dilution, TFE3 (Sigma #HPA023881), 1:200 dilution.

Incubate at 4°C 1h 0m 0s.

Aspirate antibody solution and wash cells with PBS for 0h 5m 0s.

Repeat wash.

Wash with PBS for 0h 5m 0s. (1/3)

Wash with PBS for 0h 5m 0s.(2/3)

Wash with PBS for 0h 5m 0s.(3/3)

Add filtered PBS containing 5% BSA and secondary antibodies (1:600, Alexa fluorophores 488 and 555, Invitrogen).

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

Aspirate antibody solution and wash cells.

Wash cells with PBS for 0h 5m 0s. (1/3)

Wash cells with PBS for 0h 5m 0s. (2/3)

Wash cells with PBS for 0h 5m 0s. (3/3)

Rinse coverslips in milliQ water.

Mount coverslips onto slides using ProLong Gold Antifade Mountant with DAPI (ThermoFisher P36935).

Allow slides to cure 0h 5m 0s in darkness.

Image.