The cells were fixed with 4% paraformaldehyde in PBS. After blocking with 20% AquaBlock (EastCoast Bio, North Berwick, ME, USA) for 30 min at room temperature, the cells were incubated overnight at 4°C with a guinea pig anti-INS antibody (1:100; Abcam, Tokyo, Japan) or rabbit anti-C-PEPTIDE antibody (1:200; Cell Signaling Technology, Danvers, MA, USA) and then for 1 h at room temperature with fluorescein isothiocyanate (FITC) or Alexa Fluor 647-conjugated anti-guinea pig immunoglobulin G (IgG) (FITC, 1:250 [Abcam] and Alexa Fluor 647, 1:250 [Cell Signaling Technology]), or Alexa Fluor 647-conjugated anti-rabbit IgG (1:250; Cell Signaling Technology). The cells were mounted on slides using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Peterborough, UK). The percentage of INS/C-PEPTIDE-positive cells was calculated based on the ratio of immunostaining-positive cells/DAPI-positive cells in 10 visual fields.
To identify proliferating cells, we used immunohistochemistry (IHC) to detect Ki67 in the nuclei of cells in the G1, S, G2, and M phases of the cell cycle. For this purpose, we used the Histofine Simple Stain MAX PO (R) kit (Nichire Biosciences, Tokyo, Japan) with an anti-Ki67 antibody (ab 15580) (Abcam, Cambridge, UK).
To identify proliferating cells, we used immunohistochemistry (IHC) to detect Ki67 in the nuclei of cells in the G1, S, G2, and M phases of the cell cycle. For this purpose, we used the Histofine Simple Stain MAX PO (R) kit (Nichire Biosciences, Tokyo, Japan) with an anti-Ki67 antibody (ab 15580) (Abcam, Cambridge, UK).