Rabbit anti c peptide antibody

Cells were fixed with 4% PFA in PBS for ~5 min at room temperature. After blocking with 20% AquaBlock (#PP82; East Coast Bio, North Berwick, ME, USA, https://eastcoastbio.com/) for 30 min at room temperature, cells were incubated overnight at 4°C with goat anti-Pdx1 antibody (1:100; #ab47383; Abcam, Tokyo, Japan, http://www.abcam.com/), goat anti-insulin antibody (1:100; #ab7842; Abcam), or rabbit anti-C-peptide antibody (1:100; #4593; Cell Signaling, Tokyo, Japan, http://www.cellsignal.com/). This was followed by incubation for 1 h at room temperature with Alexa Fluor 488- conjugated donkey anti-goat IgG H&L (1:200; #ab150129; Abcam), FITC-conjugated anti-goat IgG (1:250; #ab6904; Abcam), or Alexa Fluor 647-conjugated anti-rabbit IgG (1:250; #4414; Cell Signaling), respectively. A medium containing DAPI (#H-1200; Vector Laboratories, Burlingame, CA, USA, https://www.vectorlabs.com/default.aspx) was used for mounting and nuclear staining.

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).

The cells were fixed with 4% paraformaldehyde in PBS buffer. 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-insulin antibody (1:100; Abcam, Tokyo, Japan), rabbit anti-C-peptide antibody (1:200; Cell Signaling Technology, Danvers, MA, USA), goat anti-Pdx1 antiserum (1:100; R&D system, Minneapolis, MN, USA), or mouse anti-Nkx6.1 antiserum (1:100; Developmental Studies Hybridoma Bank, Iowa city, Iowa, USA) and then for 1 h at room temperature with FITC-conjugated anti-guinea pig IgG (1:250; Abcam), Alexa Fluor 647-conjugated anti-rabbit IgG (1:250; Cell Signaling Technology), NorthernLights^TM NL493-conjugated anti-goat IgG (1:200; R&D system, Minneapolis, MN, USA) or TRITC-conjugated anti-mouse IgG (1:200; Sigma-Aldrich). Mounting medium for fluorescence with DAPI (Vector Laboratories, Peterborough, UK) was used for mounting. The percentage of insulin/C-peptide-positive cells was calculated based on the ratio of immunostaining-positive cells/DAPI-positive cells in 12 visual fields.