Anti ki 67 8d5

Briefly, 1 × 10⁵ cells were seeded on 35-mm glass bottom tissue culture dishes (MatTek Corporation) in growth media. At ~ 70–80% confluence, cells were fixed, permeabilized with freeze cold methanol for 10 min, and washed in PBS. Subsequently, cells were labeled with primary antibodies: anti-phospho-Histone H3 (Ser10) (6G3) (Cell Signaling, #9706) anti-Ki-67 (8D5) (#9449, Cell Signaling). After overnight staining at 4˚C, cells were washed in PBS and stained with secondary antibody (Anti-mouse IgG (H + L), F(ab')2 Fragment [Alexa Fluor® 488 Conjugate] #4408 Cell Signaling) at room temperature. After 2 h, the cells were washed in PBS and stained with Hoechst H33342 (5 µg/mL) to visualize nuclei. Stained cells were imaged within 24 h with the Keyence BZ-X810 imaging system.

Antibodies include: anti-GSK-3β (D5C5Z, #12456), anti-phospho-GSK-3β (Ser9,5B3) (#9323) anti-Ki-67 (8D5, #9449) anti-Cleaved Caspase-3 (Asp175) (#9661) from Cell Signaling Technology (CST); anti-phospho-serine/threonine (ab15556) and anti-phospho-serine (ab9332) from Abcam; anti-Flag^® M2 from Sigma-Aldrich; anti-RARB (A1603) from ABclonal; anti-PARP1 (66520-1-Ig) and anti-His (66005-1-Ig) from Proteintech; anti-rabbit and anti-mouse secondary antibodies conjugated to horseradish peroxidase, anti-rabbit secondary antibodies conjugated to Cy3 from Invitrogen; anti-RXRα (ΔN197, sc-774; D20, sc-553), anti-RARα (C20, sc-551), anti-GFP (B-2,sc-9996), anti-c-Myc (9E10, sc-40), anti-β-actin (H196, sc-7210) and anti-GAPDH (FL-335, sc-25778) from Santa Cruz Biotechnology. Chemicals and other regents are: lipofectamin 2000 from Invitrogen; enhanced chemiluminescence (ECL), protein A/G agarose from ThermoFisher Scientific; sorafenib, BIO, SB415286 and tideglusib from Selleck Chemicals; 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 4,6-Diamidino-2-phenylindole (DAPI), 9-cis-RA, isopropyl-1-thio-b-D-galactopyranoside (IPTG) and LiCl from Sigma-Aldrich; cocktail of proteinase and phosphatase inhibitors from Roche; Dual-Luciferase Assay System Kit from Promega.

The tumor tissue was fixed in 4% paraformaldehyde. All specimens were embedded in paraffin and sliced into 10 µm thick sections. After dewaxing and hydration, the sections were incubated in 3% H₂O₂ to eliminate endogenous peroxidase activity. Then the sections were blocked with 0.5% bovine serum albumin for 1 h at room temperature. The sections were subsequently incubated overnight at 4 °C with anti-Ki-67 (8D5) (Cell Signaling Technology #9449S), anti-histone H3 (acetyl K9) (Abcam #ab10812), anti-c-Met (Cell Signaling Technology #8198S), anti-IKKα (Abcam #ab32041), and anti-Bcl-XL (Cell Signaling Technology #2764S). The sections were observed under a light microscope. TUNEL staining was performed with an in situ cell death detection kit, POD (Roche #11684817910) according to the manufacturer’s protocol. The same quantification method used for the IHC analyses was applied in the TUNEL analysis. The relative fluorescence intensity and the number of IHC-positive cells in each section were measured and quantified in NDP.view software.