Fitc conjugated anti guinea pig igg

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-insulin antibody (1:100; Abcam, Tokyo, Japan) or a mouse anti-glucagon antibody (1:200; Merck, Tokyo, Japan) and then for 1 h at room temperature with FITC-conjugated anti-guinea pig IgG (1:250; Abcam) or TRITC-conjugated anti-mouse IgG (1:250; Merck). Sections were treated with mounting medium to detect the fluorescence emitted by DAPI (Vector Laboratories, Peterborough, UK).

A guinea pig anti-insulin antibody (Abcam, Tokyo, Japan) and a FITC-conjugated anti-guinea pig IgG (Abcam) were used for immunofluorescence staining of mouse pancreatic islets. Pancreatic tissues were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). Paraffin sections were mounted on slides. After blocking with 20% AquaBlock (EastCoast Bio, North Berwick, ME, USA) for 30 min at room temperature, the sections were incubated overnight at 4 °C with a guinea pig anti-insulin antibody (1:100); then, they were incubated for 1 h at room temperature with a FITC-conjugated anti-guinea pig IgG (1:250).

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.