Biotinylated anti goat igg

Five micrometer sections were cut from the paraffin-embedded bovine CLs, dewaxed and washed in phosphate-buffered saline (PBS). Subsequently, the sections were incubated at room temperature with 3% hydrogen peroxide in distilled water for 20 min and avidin/biotin blocking solution (Vector Laboratories, Burlingame, CA, USA) for 15 min for each reagent. Then the sections were incubated with 10% normal donkey serum for 60 min at room temperature followed by goat anti-human galectin-1 antibody (1:1000; AF1152, R&D Systems, Minneapolis, MN, USA) at 4 C overnight. Control sections were incubated with PBS. After washing twice in PBS, the sections were incubated with biotinylated anti-goat IgG (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 60 min at room temperature. The reaction sites were visualized using a Vectastain ABC Elite kit (Vector Laboratories) for 60 min at room temperature and ImmPACT^® DAB (3, 3’-diaminobenzidine) Peroxidase
Substrate Kit (Vector Laboratories) for 5 min. The sections were counterstained for 2 min with hemotoxylin and observed under a light microscope (BX51, Olympus, Tokyo, Japan).