Rabbit anti b4galnt3 polyclonal antibody

Human colorectal cancer tissue sections were de-paraffinized in xylene and re-hydrated in a series of graded alcohols. After rinsing twice with PBS, the sections were then quenched the activity of endogenous peroxidase with Ultravision Hydrogen Peroxide Block (Thermo scientific, Barrington, IL) for 10 min and incubated with Ultravision Protein Block (Thermo scientific, Barrington, IL) for 10 min to reduce non-specific binding. Sections were incubated with an rabbit anti-B4GALNT3 polyclonal antibody (Sigma, St. Louis, MO, 1:100) diluted with 1% bovine serum albumin (MDBio, Inc., Taipei, Taiwan)/PBS for 16 h at 4 °C. After rinsing twice with PBS, the sections were processed using the Ultravision Quanta Detection System (Thermo scientific, Barrington, IL). Specific immuno-staining was visualized with DAB Quanto (Thermo scientific, Barrington, IL). All sections were counterstained with hematoxylin for 1 min and mounted with UltraKitt (J.T. Baker, Deventer, Holland). Negative controls were performed by replacing primary antibodies with rabbit IgG (SouthernBiotech, Birmingham, AL) at the same concentration.

B4GALNT3 proteins were detected with rabbit anti-B4GALNT3 polyclonal antibody (Sigma, St. Louis, MO). Detection of glycoproteins decorated with LacdiNAc structure was achieved by using biotinylated Wisteria floribunda agglutinin (WFA) (Vector laboratories, Burlingame, CA). For EGFR and its downstream signaling analyses, antibodies against total EGFR, EGFR pY 845, EGFR pY1068, p-AKT, AKT, p-ERK1/2, ERK1/2 (Cell Signaling Technology, Danvers, MA), and GAPDH (BD Biosciences, San Jose, CA) were used. Signals on Western blots were quantified by ImageJ 1.42q software (Wayne Rasband, NIH, Bethesda, MA).