Mouse anti collagen 1

hPIs in suspension and encapsulated in hydrogel maintained in culture for 1, 14 and 28 days were fixed in paraformaldehyde (PFA) 2% and 4% (w/v in PBS) and cryosectioned at 50 µm-thick via Cryostat (Histo-Line Laboratories). Sections were permeabilized with 0.3% Triton X-100 for 10 min at 4°C and blocked with 10% normal goat serum (NGS, Gibco) for 1 h at room temperature. The following primary antibodies were used: rabbit anti-insulin (1:300, ThermoFisher), mouse anti-chromogranin (1:100, ThermoFisher), mouse anti-glucagon (1:8000, Sigma-Aldrich), rabbit anti-Ki67 (1:750. Novus Biologicals), rabbit anti-vWF (1:500, DakoCytomation), mouse anti-fibroblast (1:200, Acris Antibodies), rabbit anti-collagen IV (1:100, Cedarlane), mouse anti-collagen I (1:2000, Sigma-Aldrich), rabbit anti-laminin (1:30, Sigma-Aldrich). To reveal primary antibodies, the following secondary antibodies were used: goat anti-rabbit Cy3 (1:1,000, Jackson), goat anti-mouse Cy3 (1:1,000, Jackson), goat anti-rabbit Alexa 488 (1:1,000, Invitrogen) and goat anti-mouse Alexa 488 (1:1,000, Invitrogen). Cell nuclei are stained with HOECHST 33342 (1:500, Molecular Probes).

Immunofluorescence staining was performed according to a previous report^{67 (link)}. Briefly, the cells were fixed in 10% buffered formalin for 30 min and washed with PBS. Non-specific binding was blocked using 10% horse serum. The cells were stained with primary antibodies at 4 °C overnight. The cells were then incubated with biotinylated secondary antibodies (Invitrogen) for 30 min and subsequently stained with Strep-FITC (Sigma). The nuclei were counterstained with DAPI (Sigma). Protein expression was visualized under a fluorescent microscope. The primary antibodies used were mouse anti-collagen I (C2456, Sigma), anti-OPN (AB1870, Merck Ltd.), and anti-RUNX2 (8486, Cell Signaling Technology).

Cells were fixed with 4% buffered formalin for 10 min. Horse serum (2% v/v) was used to inhibit a non-specific binding. Cells were stained with mouse anti-collagen I (C2456, Sigma) at 4 °C overnight and subsequently incubated with biotinylated anti-mouse antibodies (Invitrogen) for 40 min. The targeted protein expression was visualized by stained with Strep-FITC (Sigma). DAPI (TOCRIS bioscience) was employed for nuclei staining.