Optimized Immunohistochemistry Protocol for OPN and E-Cadherin

Formalin-fixed, paraffin embedded tissues were sectioned onto ProbeOn Plus microscope slides (Fisher Scientific) and immunohistochemistry was performed as described [20 (link)]. For immunohistochemistry studies, anti-OPN (final dilution: 1:200, Maine Biotechnology Services Catalog #MAB197P, mouse monoclonal antibody raised against recombinant OPN of human origin, [21 (link)]) or anti-e-Cadherin (final dilution: 1:100, Cell Signaling Technologies Catalog #3195T) were used and were detected using the Vector ABC kit (Vector Laboratories, Burlingame, CA, USA). For immunofluorescence, anti-OPN (final dilution: 1:50, Clone AKm2A1, Santa Cruz Catalog # sc-21742, mouse monoclonal antibody raised against recombinant OPN of mouse origin, references with validation available on manufacturer’s datasheet) was used and was detected using a goat anti-mouse IgG AF549 conjugated secondary antibody (final dilution: 1:1000, Invitrogen Catalog # A11032, polyclonal, references with validation available on manufacturer’s datasheet). Nuclei were counterstained with DAPI (Invitrogen). Images were captured with identical exposure and gain using a Nikon Eclipse Ni microscope.