Ab78547

IHC staining and IF staining were performed using standard procedures [39 (link)]. For immunostaining, slices were covered in 0.01 M sodium citrate buffer at 65 °C for 10 h for antigen recovery, subjected to 0.5% Triton-X 100 for 20 min to break the cell membranes, and then incubated with 10% FBS for 1 h to block non-specific binding sites. Incubation with primary antibody was performed according to the instructions, and the used primary antibodies included pTBK1 (5483, CST), CD68 (ab955, Abcam), Tommo20 (ab78547, Abcam), dsDNA (ab27156, Abcam), and Col2 (ab34712, Abcam).
For IHC staining, sections were subjected to corresponding horseradish peroxidase (HRP)-labeled secondary antibodies for 1 h. Diaminobenzidine (DAB) solution was utilized to visualize the target protein. The images were then captured with a microscope (VS200, Olympus, Japan). For IF staining, slices were incubated with fluorescent secondary antibody at 37 °C for 1 h. The slices were then stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed with a confocal fluorescence microscope (IX83-FV3000, Olympus, Japan). Quantitative analysis was performed with ImageJ. The proportion of positively stained cells was counted and averaged on at least 3 sections for analysis. Approximately 20 immunopositively stained cells were counted on each section.

For all cell types and conditions, cells were fixed with 1% glutaraldehyde (Electron Microscopy Sciences, 16020) for 10 min and subsequently washed three times with sodium borohydride (Fisher Chemical, S678; 1 mg/ml, 15 min interval) and then permeabilized with 0.25% Triton X-100 for 10 min. After permeabilization, they were washed thrice again with PBS and incubated in blocking buffer (10% NCS in PBS) for 30 min. The cells were then incubated with anti-Tom20 (ab78547 1:500; Abcam) antibody prepared in 0.1% blocking solution for 90 min. Following PBS washes, the cells were incubated with secondary antibody against Tom20 (Alexa Fluor 488-coupled anti-rabbit; #A11037; 1:200; Invitrogen) mixed with TRITC-phalloidin (P1951 1:400; Sigma-Aldrich), 1× DAPI (D9542; Sigma-Aldrich), and incubated for 60 min. The cells were then washed with PBS, resuspended in 2 ml PBS, and imaged on the same or following day.

For immunofluorescence analyses of colocalization, cells were fixed for 30 min at room temperature with 4% paraformaldehyde, and then were permeabilized with 0.2% Triton X-100. Subsequently, the cells were incubated in a blocking solution of phosphate-buffered saline/Tween-20 with 10% goat serum and 3% bovine serum albumin for 1 h at room temperature [34 ]. The samples were then incubated with the primary antibodies overnight at 4 °C, and with appropriate fluorescent secondary antibodies for 3 h at room temperature. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Images were captured with a laser-scanning confocal microscope (LSM700; Carl Zeiss Inc., Oberkochen, Germany). Primary antibodies for immunoblotting were as follows: Gr1 (1:500, Abcam, #ab25377), troponin T (1:500, Abcam, #ab8295), CD31 (1:500, Abcam, #ab222783), VE-Cadherin (1:500, Abcam, #ab33168), Tom-20 (1:500, Abcam, # ab78547). Mitophagy was determined through mt-Kemia as our previously described [22 (link)].

Standard FACS analysis was performed with BD Fortessa. Extracellular mitochondria were analyzed using an adapted method as previously reported (42 (link)). Briefly, mito-DsRed–labeled NPCs were treated with FNZ for 3 days (the culture medium was replaced freshly at day 2). The culture supernatant, harvested at day 3, was spun at 500g for 5 min and passed through 0.45-μm filters to remove dead cells. The resultant supernatant was used for FACS analysis. Cell surface staining of TOM20 (ab78547, Abcam) was performed as described previously (34 (link)). Briefly, NPCs were treated with FNZ for 1 day, followed by cell collection (without fixation) and staining with a TOM20 antibody and secondary antibody. Gating was performed on the basis of an antibody-free control.