8 well chamber slides for immunofluorescence

First, 3 × 10⁴ HEK293 cells/well were plated in 8-well chamber slides for immunofluorescence (Ibidi, Gräfelfing, Germany). Transient transfection was performed as described above, and cells were washed and fixed in Methanol (Sigma-Aldrich) for 5 min at −20 °C and blocked in blocking solution (Phosphate Buffered Saline (PBS, Sigma-Aldrich) containing 3% Bovine Serum Albumin (BSA, Sigma-Aldrich). Rabbit anti-B7-H4 primary antibody (1/200 in blocking solution) was incubated overnight at 4 ℃ in a wet chamber. Subsequently, cells were washed three times with PBS-BSA for 10 min prior to incubation with anti-rabbit FITC secondary antibody (1/100) for 1 h in a wet chamber and darkness at room temperature. Cells were washed and mounted in a Mounting Medium with DAPI (Abcam, Cambridge, UK) and visualized in a confocal microscope (ZEISS LSM880 Airyscan, Jena, Germany). For quantitation of B7-H4 subcellular distribution, at least 50 positive cells were scored. Cells were rated as membrane staining (M) or membrane/cytoplasm (M/C). Nuclei were identified by DAPI staining.

3x10⁴ COS-7 cells per well were plated in 8-well chamber slides for immunofluorescence (Ibidi, Gräfelfing, Germany). Transient transfection was performed as described above. Cells were washed and mitochondria were stained with Mitotracker™ Red CMXRos following manufacturer’s instructions (250 nM, 20 min) (ThermoFisher). before they were fixed in methanol for 5 min at -20°C and blocked in blocking solution (Phosphate Buffered Saline (PBS) containing 3% Bovine Serum Albumin (BSA). Mouse anti-Flag primary antibody (1/100 in blocking solution) was incubated overnight at 4°C in a wet chamber. Subsequently, cells were washed three times with PBS-BSA for 10 min prior to incubation with anti-mouse FITC secondary antibody (1/100) for 1 h in a wet chamber and darkness at room temperature. Cells were washed and mounted in Mounting Medium with DAPI (4’6-diamidino-2-phenylindole) (Abcam) and visualized by standard [NIKON ECLIPSE TE2000 (Nikon, Tokyo, Japan)] or confocal microscopy [ZEISS LSM880 AIRYSCAN (Zeiss, Jena, Germany)].