Ab1893

Animals were administered with BrdU (10 mg/mL in physiological saline; Sigma-Aldrich) twice per day by intraperitoneal injection at a dose of 350 mg per kilogram body weight, 3 days before sacrifice. After ether anesthesia, animals were transcardially perfused with 50 ml normal saline and 50 mL 4% PFA. The brains were fixed in 4% PFA overnight at 4°C, then cryoprotected in 30% sucrose and embedded in embedding medium (Tissue-Tek; Sakura Finetek, Torrance, CA). Transverse sections were cut using a cryostat (10 µm) (Leica, Wetzlar, Germany). For immunofluorescence staining, sections were re-hydrated (three PBS washes and 2 mol/L HCl for 1 h at RT). After four PBS washes, sections were treated with blocking solution (PBST and 10% goat serum) for 1 h at RT, then incubated in sheep-anti-BrdU antibody (ab1893, Abcam, Cambridge, MA) solution (1:200 in blocking solution) at 4°C overnight. After three PBS washes, sections were incubated in Alexa Fluor 488 donkey-anti-sheep antibody (1:300, Invitrogen/Molecular probe) for 2 h at RT. Following three PBST washes, the nuclei were stained with Hoechst (10 µg/mL) at RT for 20 min. The sections were mounted with GVA mounting medium. The number of BrdU-positive cells was determined from five consecutive sections (Wojtowicz and Kee 2006 (link)).

NSCLC cells were prepared into single-cell suspension and planted on slides positioned in 12-well plates (1 × 10⁵ cells/well). After NSCLC cells adhered to the bottom of the wells, 10 μmol/L of BrdU solution was added, and the cells were incubated for 4 h at 37°C. The solution was then discarded and the cells were rinsed 3 times with PBS. Subsequently, 70% ethyl alcohol was added, and the cells were fixed at 4°C for 10 min, and after 70% ethyl alcohol was discarded, the cells were rinsed using PBS 3 times. Then, 2 mol/L of HCl was added and maintained at 37°C for 40 min to denature DNA. Thereafter, HCI was discarded and the cells were rinsed with PBS 3 times. After 1% FBS was supplemented, the cells were blocked for 1 h. Then, the cells were rinsed with PBS again, and anti-BrdU monoclonal antibody (Abcam, ab1893, 1:300) was added to each well, and the cells were incubated overnight at 4°C. On the next day, Cy3 labelled goat anti-mouse fluorescent secondary antibody was added and incubated with cells for 2 h. Ultimately, the nuclei were counterstained with DAPI and the cells were observed under fluorescence microscope. Four fields were randomly taken from each slide, and the number of BrdU-positive cells was counted. Cell multiplication rate = the number of BrdU staining positive cells/total number of cells×100%.