Anti ki67 antibody

All animal experiments were carried out according to the Institutional Ethical Guidelines for Animal Experimentation at Osaka University and reported in accordance with Animal Research: Reporting of In Vivo Experiments guidelines [21 ].
For the development of a xenograft model, 2.0 × 10⁶ HEC1 cells were injected into Institute of Cancer Research nu/nu mice subcutaneously. Tumor volumes were evaluated twice a week [length × width² × 0.5]. When the mean tumor volume reached approximately 100 mm³, the mice were randomized into two groups (5 mice per group) and received antibody therapy: an isotype control mouse IgG2a antibody (control Ab) (Sigma Aldrich, MO, USA) or chimeric chicken–mouse anti-LSR mAb (#1–25; Pharmafoods). These antibodies were administrated intraperitoneally at a dose of 200 μg/body twice a week for 3 weeks. IHC staining in resected tumors was performed using anti-phospho-ERK1/2 antibody (Thr202/Tyr204) (#4370), anti-Ki-67 antibody (#9027), or anti-cleaved caspase-3 antibody (#9661) from Cell Signaling Technology (MA, USA). IHC-positive cells were imaged (400 X) and counted in three microscopic fields per lesion using a fluorescence microscope (BZ-X710) and BZ-X Analyzer software from Keyence (Osaka, Japan).

Fresh frozen liver tissue was sectioned at 8 μm and stained using previously described methods35 (link). Anti-YAP antibody and anti-Ki67 antibody (Cell Signaling, Beverly, MA) were used for primary incubation. For secondary antibody incubation we utilized Cy3-conjugated goat anti-rabbit and FITC-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). The data was analyzed using open source software from the National Institutes of Health (ImageJ).

Xenograft tumors were collected on the day after the last dosing; tumor fragments were fixed with 10% formalin and embedded in paraffin, except for LI0334 tumor fragments, which were frozen in OCT compound and the tissue sections were fixed with cold acetone. Staining for endothelial cells with anti‐CD31 antibody and microvessel density (MVD) measurement were performed as described previously 25. Briefly, tissue sections were stained with anti‐CD31 antibody (Dianova, Hamburg, Germany) and the slides were scanned using the Aprio ScanScope XT system. Five regions of interest (ROIs, each 500 × 500 μm) with the highest densities of CD31‐stained microvessels were selected manually, and the number of microvessels in each ROI was measured.
Ki‐67‐positive cells were stained with anti‐Ki‐67 antibody (Cell Signaling Technology) and detected using Envision+ Single Reagents (Dako, Agilent Technologies, Santa Clara, CA). The percentage of Ki‐67 positive nuclei was quantified using HALO software (Indica Labs, Corrales, NM).

Harvested tumors were fixed in 4% PFA for 24 h at room temperature, followed by permeabilization with PBST for 20 min and inactivation of endogenous peroxidases by incubating tissues in 0.3% H₂O₂ for 20 min. After washing, tissues were blocked with 5% normal goat serum (Invitrogen, USA) for 30 min and incubated with anti-Ki-67 antibody (Cell Signaling Technology, USA) overnight at 4 °C. Subsequently, tissues were rinsed and probed with a biotin-labeled secondary antibody for 30 min and washed. After washing, the specimens were developed with 0.05% DAB (Sigma-Aldrich, Oakville, ON, Canada) and 0.03% H2O2, and counterstained with hematoxylin, dehydrated in increasing ethanol concentrations, cleared with xylene. Images of stained specimens were captured using an Olympus BX51 microscope. Digital images were analyzed using Image-Pro Plus 6.0 software.

Cisplatin (cat. no. HY-17394) and PI3K/AKT inhibitor (cat. no. HY-10108) were purchased from MedChemExpress. The antibodies used for western blot and immunohistochemistry were as follows: anti-DKK1 antibody (cat. no. ab307367), anti-Phospho-PI3K antibody (cat. no. 4228) were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology (Danvers, MA, USA); anti-Ki67 antibody (cat. no. ab15580) was purchased from Abcam; anti-vimentin antibody (cat. no. 60330-1-lg), anti-N-cadherin antibody (cat. no. 22018-1-AP), anti-E-cadherin antibody (cat. no. 60335-1-lg), anti-GAPDH antibody (cat. no. 60004-1-Ig), anti-PI3K antibody (cat. no. 60225-1-Ig), anti-AKT antibody (cat. no. 60203-2-Ig), anti-Phospho-AKT (cat. no. 28731-1-AP) were purchased from Proteintech Group (Rosemont, IL, USA). HRP-linked secondary antibody anti-rabbit lgG (cat. no. 7074) and anti-mouse lgG (cat. no. 7076) were obtained from Cell Signaling Technology.

Skin equivalents were irradiated with IRA (800 J cm⁻² day⁻¹) for up to 5 consecutive days. Each tissue was fixed with 10% formalin and embedded in paraffin, and cross‐sections (5–7μm thick) were stained with hematoxylin‐eosin to measure the thicknesses of the epidermal cell layer and stratum corneum. The epidermal cell layer was defined as the distance from the top of the granular layer to the bottom of the basal layer. To examine the expression of Ki‐67, specimens were treated with anti‐Ki‐67 antibody (Cell Signaling Technology, #9449, 1:400) for 1 h at room temperature. The number of Ki‐67‐positive cells in the microscopic field at × 10 magnification of each tissue section was counted (5 sections per group). Bright field images were acquired using a fluorescence microscope (BZ‐X700; Keyence).

For BrdU assay, ARK2 cells were treated with BrdU for 2 h after transfecting with wild-type LSD1 or LSD1 S707A/S711A for 48 h. DNA synthesis activity was measured with BrdU ELISA kit (Roche applied science). For Ki67 staining, after tranfecting with wild-type LSD1 or LSD1 (S707A/S711A) in ARK2 cells for 48 h^{8 (link)}, Ki67 was stained with an anti-Ki67 antibody (Cell Signaling Technology).