Anti ki67 antibody ab16667

Antibodies against DNMT1 (EM1901-83), BrdU (RT1081), mTOR (ET1608-5), GSK3β (ET1607-71), p-GSK3β (Ser9, ET1607-60) and PKM2 (ER1802-70) were obtained from HuaBio. Antibodies against Ki67 (9449 T, for immunostaining), PI3K (4249 T), p-mTOR (Ser2448, 5536 T), AKT (4691 T), p-AKT (Ser473, 4060 T), cleaved caspase-3 (CC3, 9664S) and p-Rb (Ser807/811, 8516S) were obtained from Cell Signaling Technology. Anti-Ki67 antibody (ab16667, for immunoblotting), anti-CDK1 (phospho T161) + CDK2/CDK3 (phospho T160) antibody (ab201008) and anti-phosphofructokinase antibody (PFK, ab154804) were obtained from Abcam, while an antibody against 5-mC (A-1014) from Epigentek. All the inhibitors and activators used were from MCE, including BEZ235 (HY-50673) for PI3K inhibition, 740Y-P (HY-P0175) for PI3K activation, AT7519 (HY-50943) for CDK2 inhibition, and GSK-3484862 (HY-135146) and GSK-3685032 (HY-139664) for DNMT1 inhibition.

Prepared 7 µm skin sections were incubated in citrate buffer at 60°C overnight (antigen retrieval). The endogenous peroxidase activity was blocked by incubation in 1% hydrogen peroxide in PBS for 15 minutes. The following rabbit polyclonal anti-mouse antibodies were used: anti-involucrin (PRB-140C), anti-filaggrin (PRB-417P), anti-loricrin (PRB-145P), anti-keratin 5 (PRB-160P), anti-keratin 6 (PRB-169P), anti-keratin 10 (PRB-159P) (all from Covance, Princeton, NJ, USA). The rabbit monoclonal anti-Ki67 antibody (ab16667) was purchased from Abcam (Cambridge, UK). For immunodetection the 3,3′-diaminobenzidine (DAB) Detection Kit (USA™ Ultra Streptavidin Detection System, Covance, SIG-32232) was used according to the manufacturer’s instructions. For immunofluorescence, we followed the Immunofluorescence Standard Protocol published by the Cell Signaling Technology company. The results were documented using microscope (Eclipse 80i, Nikon, Tokyo, Japan) with digital camera.

hADSCs were seeded into 6-well tissue culture plates at a density of 5 × 10⁴ cells/well. After green OLED irradiation, cells were fixed with 4% paraformaldehyde. These fixed cells were blocked with 10% (v/v) normal goat serum (Jackson Immuno Research Laboratories, West Grove, PA, USA) and 0.6% (v/v) Triton X-100 (Sigma-Aldrich) in PBS at room temperature for 1 h. Cells were then immunofluorescence stained with anti-KI67 antibody (ab16667; Abcam) and fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG (H + L) (Jackson Immuno Research Laboratories). DAPI was used as a counterstain. Stained cells were examined using a fluorescence microscope (DFC 3000 G).