Click it edu alexa fluor 647 or 488 imaging kit

To detect EC proliferation in adult livers, 20 μg per g body weight EdU (Invitrogen, A10044) was injected intraperitoneally into adult mice 5 h before dissection. Livers were isolated for cryosection analysis. EdU signals were detected with the Click-iT EdU Alexa Fluor 647 or 488 Imaging Kit (Invitrogen, C10340 or C10337). In brief, after all other primary and secondary antibody incubations, samples were washed according to the immunofluorescence staining procEdUre and then incubated with Click-iT EdU reaction cocktail for 40 min, followed by DAPI counterstaining.

T47D or SUM190-PT cells were plated at a density of 3 × 10⁴ cells per square centimetre on dishes and cultured at 37 °C for 48 h. The cells were starved of serum, cultured with RPMI1640 or Ham’s F12 containing 0.5% fatty acid-free BSA at 37 °C in the absence or presence of the inhibitors for 24 h and then treated with 10 μM EdU at 37 °C for 12 h. After washout of EdU-containing media, EdU-treated cells were additionally cultured with serum-free medium in the absence or presence of the inhibitors at 37 °C for 12 h and fixed using 4% paraformaldehyde diluted in PBS. The signal for EdU and nuclei in the cells was determined by the Click-iT EdU Alexa Fluor 488 or 647 Imaging Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. The samples were counted by fluorescence microscopic examination. The images were acquired using a BZ-X710 microscope (KEYENCE CORPORATION, Osaka, Japan) with a CFI Plan Apo λ 20 × /0.75 numerical aperture objective lens (Nikon, Inc., Tokyo, Japan) in 1,920 × 1,440 pixels. The displayed images were applied into maximum signal intensity projection from around 5 confocal images collected at a 2 μm step along the z-axis at room temperature under the control of BZ-X Viewer software (KEYENCE CORPORATION). The images were processed using ImageJ 1.48 v 32-bit software.

To label proliferating cells in the TM at the time of tissue harvest, mice were injected while awake 2-h prior to takedown with EdU (Carbosynth Limited) was resuspended at 5 mg/mL in saline and passed through a 0.22 μm filter. Mice were each injected with 1 mg (200 μL) by intraperitoneal (IP) injection. To fully label and track the proliferating cell population in the TM over time, EdU was administered continuously, first by IP injection at the start of the experiment and then via supplementation in the drinking water at a concentration of 0.5 mg/mL with 1% sucrose that had been filtered using a 0.2-μm filter. The water supply was changed every three days with care taken to protect the water bottles from light. TMs were dissected and processed as described for IF. The Click-iT EdU Alexa Fluor 488 or 647 Imaging Kit (ThermoFisher Scientific) was used for EdU detection. For combined EdU and protein detection, the IF protocol was followed after EdU detection, starting from the blocking step. Quantification of EdU-labeled cells was performed using Fiji (ImageJ).