Mouse anti tim23

Preparation of cells for immunofluorescence was carried as previously described⁶⁵ with several modifications. Briefly, MEL cells were attached to poly-L-lysine coated coverslips (BD Bioscience) by incubating undifferentiated cells on coverslips for 24 hours at 37°C in 5% CO₂. Cells were fixed in 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100 for 20 minutes and blocked with 5% bovine serum albumin for 1 hour. Cells were incubated with rabbit anti-PGRMC1 (Sigma) diluted 1:100 and mouse anti-TIM23 (BD Biosciences) at a dilution of 1:200 overnight. Incubation with AlexaFluor 488 goat anti-rabbit IgG (H+L) (Life Technologies, Carlsbad, CA) and AlexaFluor 633 goat anti-mouse IgG (H+L) (Life Technologies) was carried out for 1 hour. Finally cells were counterstained with 300 mM DAPI nucleic acid stain (Thermo Fisher Scientific) for 5 minutes prior to mounting with ProLong Gold antifade (Life Technologies). Cells were washed with phosphate buffered saline (PBS) three times between each step in the process, reagents were diluted in PBS and all incubations were carried out at room temperature. Cells were imaged using a Zeiss LSM 710 Inverted Confocal microscope (Zeiss, Thornwood, NY) using a 100X oil immersion objective and images processed using Zen (Zeiss) software.