Fitc conjugated anti mouse antibody

Fixation of flower buds, slide preparations of PMCs, and fluorescence in situ hybridization (FISH) were carried out according to Sánchez-Morán et al. (2001 (link)). The DNA probes used comprise ribosomal DNA 5S and 45S loci to identify individual bivalents (Gerlach and Bedbrook, 1979 (link); Campell et al., 1992 (link)), and a 180 bp repeat sequence (pAL1) to detect centromeres (Martínez-Zapater et al., 1986 (link)). These probes were labeled with either biotin-dUTP or digoxygenin-dUTP, using a nick translation kit (Roche). For 5-methylcytosine immunodetection, the slide preparations were dried at 60°C (30 min), fixed in 1% formaldehyde (10 min), dehydrated through an ethanol series (70, 90, 100%) and then dried at room temperature. After denaturation using HB50 at 80°C (2 min), slides were rinsed in ice-cold 2×SSC and incubated in 1% BSA (1 h). The preparations were then washed three times in PBS and one in TNT (5 min each). For the immunodetection, slides were incubated with anti-5-methylcytosine raised in mouse (1:50, Eurogenetec) during 30 min and washed three times in TNT (5 min). Finally, a FITC-conjugated anti-mouse antibody (1:500, Abcam) was used.

Primary lung fibroblasts were cultured in a dish at 37 °C, and the mitochondria were stained with 100 nM MitoTracker Red (Invitrogen) for 20 min. Then, the cells were fixed with 4% (v/v) paraformaldehyde (PFA) for 45 min, permeabilized in ice-cold buffer (0.1% [w/v] sodium citrate and 0.1% [v/v] Triton X-100 in distilled water) for 3 min, blocked with Dulbecco’s phosphate-buffered saline with 1% (w/v) BSA for 1 h; and incubated with an anti-OAT antibody (1:200; Santa Cruz, #SC-374243) overnight at 4 °C. After washing, the cells were incubated with a fluorescein isothiocyanate (FITC)-conjugated anti‐mouse antibody (1:1000; Abcam, #ab6785) for 1 h at room temperature and mounted in fluorescence medium containing 4ʹ,6-diamidino-2-phenylindole (DAPI; Abcam). To measure the expression of TGF-β1 in BLM-treated OAT-knockdown cells, the cells were subjected to double immunofluorescence staining for TGF-β1 (1:100; Thermo Fisher Scientific, #MA5-15065) and OAT (1:100). FITC-conjugated donkey anti-rabbit IgG (1:1000; Abcam, #ab150061) and PE-conjugated goat anti-mouse IgG (1:1000; Abcam, #ab97024) served as the secondary antibodies. All the stained images were captured using a Leica DMi8 confocal laser microscope (Leica Microsystems; Wetzlar, Germany).

Breast cancer cells were fixed in 4% paraformaldehyde and then blocked in PBS (3% bovine serum albumin). Cells were stained with the anti-CD73 antibody (1D7, 3F7 and R1107) or IgG at a dilution of 1:100 and then washed and incubated with a FITC-conjugated anti-mouse antibody (1:100, Abcam, UK) in PBS for 45 min. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. A confocal microscope (Olympus, Tokyo, Japan) was used for imaging.
Flow cytometry was used to analyze the antibody binding specificity. The cells were trypsinized and washed twice with PBS on ice. After centrifugation for 5 min at 0.4 g, the cell pellets were subsequently resuspended in a blocking buffer (PBS with 5% FBS and 3% BSA). The cells were stained with the anti-CD73 antibody or IgG (1:100) for 1 h at 4 °C. Next, the cells were washed with PBS and incubated with a secondary antibody (1:100) for 40 min. The flow cytometry was performed with a FACSCalibur flow cytometer (BD, San Jose, CA, USA).