Immunohistochemical Analysis of Transplanted Islet Cells

Generated cells and extirpated organs were prepared and stained by methods already reported^{12 (link)}. Briefly, generated IPCs were fixed using a cell fixing kit (Funakoshi), recipient mice were sacrificed and IPCs bearing kidneys were extirpated. 4 μm thick paraffin embedded sections were incubated with primary antibodies against insulin (aa287–299, LS-B129; LSBio) at a dilution of 1:100 in phosphate-buffered saline (PBS), anti-HLA class I (ab70328; abcam) 1:100 in PBS, C-peptide (bs-0274R, Funakoshi) 1:100 in PBS and pancreatic and duodenal homeobox 1 (PDX-1, ab47308;abcam) 1:200 in PBS for 1 h at room temperature after deparaffinization and antigen retrieval. Slides were then incubated with biotinylated secondary antibody, followed by treatment with a streptavidin-biotin-horseradish peroxidase complex. Positive staining was visualized with diaminobenzidine and cell nuclei were counterstained with Mayer’s haematoxylin.