For cell immunofluorescence, 2 × 104 cells were seeded on each confocal dish and cultured for 1 day. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After permeabilizing with 0.1% Triton X-100 or cold digitonin (Abcam) for 10 min and blocking with normal goat serum for 30 min, samples were incubated with primary antibodies overnight at 4 °C, including anti-TOM20 (clone F-10, Santa Cruz Biotechnology; BM4366, Boster), anti-PD-L1 (clone EPR19759, Abcam; LS-C754760, LifeSpan BioSciences; 2B11D11, Proteintech), anti-LAMP1 (clone D2D11, Cell Signaling Technology; clone 1D4B, Santa Cruz Biotechnology), anti-TGN46 (clone 1F6D5, Proteintech), anti-Calnexin (clone 2A2C6, Proteintech). The cells were washed with PBS-T for three times and incubated with secondary antibodies (Alexa Fluor Plus 555, Alexa Fluor Plus 488, Alexa Fluor Plus 647, Invitrogen) at room temperature for 1 h. Antifade Mounting Medium with DAPI (Beyotime) was used to mount the samples. The confocal images were then taken on Zeiss LSM 900 and Airyscan 2 confocal laser scanning microscope and analyzed with ZEN 2.3 software.
For MitoTracker staining, cells were incubated with MitoTracker Deep Red dyes in complete medium at 37 °C for 30 min, then were removed of excess unbound dyes by washing twice.
For tissue immunofluorescence, paraffin sections of patients with TNBC were baked at 60 °C for 30 min followed by deparaffinization and rehydration using xylene and graded ethanol. After EDTA (pH 8.0) antigen retrieval and endogenous peroxidase blockade as IHC staining methods mentioned above, the samples were permeabilized with 0.3% Triton for 30 min and blocked with normal goat serum for 30 min at room temperature. Primary antibodies targeting PD-L1, LAMP1 and TOM20 were incubated with samples overnight at 4 °C. After secondary antibody incubation for 1 h and DAPI incubation for 15 min, 0.3% Sudan black-B solution was used to quench spontaneous fluorescence for 20 min at room temperature followed by washing twice. The confocal images were then taken and analyzed.
For MitoTracker staining, cells were incubated with MitoTracker Deep Red dyes in complete medium at 37 °C for 30 min, then were removed of excess unbound dyes by washing twice.
For tissue immunofluorescence, paraffin sections of patients with TNBC were baked at 60 °C for 30 min followed by deparaffinization and rehydration using xylene and graded ethanol. After EDTA (pH 8.0) antigen retrieval and endogenous peroxidase blockade as IHC staining methods mentioned above, the samples were permeabilized with 0.3% Triton for 30 min and blocked with normal goat serum for 30 min at room temperature. Primary antibodies targeting PD-L1, LAMP1 and TOM20 were incubated with samples overnight at 4 °C. After secondary antibody incubation for 1 h and DAPI incubation for 15 min, 0.3% Sudan black-B solution was used to quench spontaneous fluorescence for 20 min at room temperature followed by washing twice. The confocal images were then taken and analyzed.
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