Cas block buffer

Immunohistochemical staining for FoxL1 was performed using the antibodies described previously (Aoki et al., 2016 (link)). Briefly, sections were deparaffinized in xylene and rehydrated in ethanol, and washed in 1× PBS for 5 min, followed by 1× PBS-T (0.25% Triton x-100) for 15 min. Antigen retrieval was performed using a Tris-EDTA antigen retrieval buffer (pH 9.0), quenched for 15 min with 3% hydrogen peroxide, blocked in Cas block buffer (Life Technologies) for 1 h, and incubated overnight with primary antibodies in 4 °C. The following day, the sections were incubated with Avidin and Biotin (Vector Labs) and incubated with biotinylated guinea pig IgG antibodies (Fisher Scientific, 1:300) for 30 min. Amplification was performed by incubation with streptavidin-peroxidase conjugate (Akoya) followed by incubation of the sections with Alexa 647 conjugated to streptavidin, and finally washed and counterstained with Hoechst (Invitrogen) before imaging.

Sections were deparaffinized in xylene and rehydrated in ethanol, and washed in 1× PBS for 5 min, followed by 1× PBS-T (0.25% Triton x-100) for 15 min, and 1× PBS for 3 × 5 min. Antigen retrieval was performed using a Tris-EDTA antigen retrieval buffer (pH 9.0). Sections were incubated for 30 min in Cas block buffer (Life Technologies) and incubated with primary antibodies overnight in 4 °C. The following day, the sections were washed 3 × 5 min. in 1× PBS, incubated with secondary antibodies for 2 h at room temperature, then washed and counterstained with Hoechst (Invitrogen) before imaging. The following antibodies were used for this study: FoxL1 (made in-house, 1:1500) E-cadherin (BD Bioscience, cat. 610,181, 1:300), EpCAM (Abcam, 1:300), Ezrin (Cell Signaling, 1:300), zona occludens 1 (ThermoFisher, 1:200), atypical Phosphokinase C (Santa Cruz Biotechnology, 1:200), Laminin (Abcam, 1:300), phospho-histone H3 (BMD millipore, 1:300), and Ki67 (BD Pharmagen, 1:300). All stained sections were imaged on a Leica TCS SP8 Confocal for immunofluorescence, and a Keyence BZ-X8100 microscope for brightfield imaging.