Diaminobenzidine tetrahydrochloride dab

Tissue microarray (TMA) sections were de-waxed using xylene twice and rehydrated with 100% ethanol for 5 minutes, 95% and 80% ethanol for 5 minute each. Then rinse in PBS. Antigen retrival was performed in 10 mM, pH 6.0 sodium citrate buffer at 95-100°C for 20 mins. After cooling down to room temperature, tissue sections were rinsed with PBS once followed by blocking endogenous peroxidase with 1% H₂O₂ and blocking non-specific binding site with Power Block (BioGenex) for 5 min at room temperature each. The tissue sections were then incubated with the specific antibody against Gal-1 (Abcam) or AR-V7 (GeneTex) or E-Cad (Cell signaling) or ki-67 (Cell signaling) overnight, followed by rinsing with PBS and incubation by an biotin-conjugated goat anti-rabbit IgG (BioGenex), as the second antibody. TMA sections were then incubated with strepavidin conjugated-HRP (BioGenex) for 20 mins at room temperature. HRP activity was detected using diaminobenzidine tetrahydrochloride (DAB) as substrate (BioGenex). Nuclei were counterstained with hematoxylin (Cell signaling). The ki-67 positive cells were counted in 3 random chosen areas.

First, we reacted neutravidin (5 nmole) with LXY30-biotin in 1:2 molar ratio for 20 min. After gluing the OB2C beads onto the 12-well culture plate with 80% dimethylformamide, mixture of neutravidin and LXY30-biotin was added onto the beads-glued wells. After 20 min of incubation, SKOV3 cells (5 × 10⁵) were then seeded onto the well and incubated at 37°C for 20 minutes. The unbound cells were removed by gentle washing with PBS. Cellbound beads were incubated at 37°C for another 24 hours, followed by fixation in 4% paraformaldehyde for 20 min. For ICC assay on beads, non-specific protein binding was blocked by adding 5% BSA and cell membrane was permeabilized with 0.5% Triton X-100. We used rabbit anti-human cleaved caspase 3 (Cell Signaling Technology) as the primary antibody. Beads were incubated with primary antibody (1:100 in PBS) for overnight at 4°C. After washing with PBS, beads were then incubated with the secondary antibody, an HRP-conjugated goat anti-rabbit IgG, for 1 hour at room temperature. HRP activity was finally detected using diaminobenzidine tetrahydrochloride (DAB) as a substrate for 3 min according to the manufacturer’s instructions (Biogenex).