Mouse anti pig c peptide

Left kidneys of the recipient mice were dissected out under general anesthesia and fixed using 10% formalin. Then, the kidney was embedded in paraffin. Three-micrometer-thick sections were either stained with hematoxylin and eosin (HE) or subjected to immunohistochemistry of porcine C-peptide to identify porcine islets, CK-19 to detect pancreatic ductal components, and swine leukocyte antigen class I (SLA I) to detect porcine-derived tissue. The primary antibodies were mouse anti-pig C-peptide (1:200; Cloud-Clone Corp. MAA447Po21, Katy, TX), rabbit anti-KRT19/CK19/cytokeratin 19 polyclonal (1:100; LSBio. LS-B13606-50, Shirley, MA), and mouse anti-pig SLA I (1:100; Bio-Rad Laboratories Inc., MCA2261PE, Hercules, CA) antibodies. After incubation with a primary antibody, donkey anti-mouse IgG (H + L) Alexa 488 (1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA,) or Cy3-conjugated goat anti-rabbit (1:100; Jackson ImmunoResearch Laboratories, Inc.) was used as a secondary antibody. Nuclear staining was performed using 4′,6-diamidino-2-phenylindole (DAPI). Histological images were obtained under the BZ-X700 microscope.

The left kidneys of recipient mice were dissected following euthanasia and the transplanted islets were evaluated. Three-µm-thick sections were either stained with hematoxylin and eosin (HE) or subjected to immunohistochemistry (for insulin to identify islets, for von Willebrand factor (vWF) to identify vessels, for porcine C-peptide to identify porcine islets, and for mouse C-peptide to identify mouse islets. The primary antibodies used were mouse anti-pig C-peptide (1:200; Cloud-Clone Corp. MAA447Po21, Katy, TX, USA), mouse anti-mouse C-peptide (1:500; Novus Biologicals NBP1-05433, Centennial, CO, USA) and rabbit anti-vWF antibody (1:100; Abcam, Cambridge, UK). After incubation with primary antibody, donkey anti-mouse IgG (H + L) Alexa488 (1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and Cy3-conjugated goat anti-rabbit (1:100; Jackson ImmunoResearch Laboratories, Inc.) were used as secondary antibodies. Nuclear staining was performed using 4′,6-diamidino-2-phenylindole (DAPI). Histological images were obtained using a BZ-X700 microscope (Keyence, Itasca, IL, USA).