Rabbit anti glucagon

Paraffin sections of the graft bearing kidney were stained with the following antibodies (Ab): guinea pig anti-insulin (1:400, Agilent-Dako), rabbit anti-glucagon (1:100, Cell Signaling), rabbit anti-somatostatin (1:50, Agilent-Dako), rabbit anti-pancreatic polypeptide (PP) (1:5000, Proteintech) and rabbit anti-CD31 (1:100, Cell Signaling). Secondary antibodies used were HRP- or alkaline phosphatase-conjugated anti-guinea pig IgG (Agilent-Dako) and anti-rabbit IgG (Vector Laboratories, California, United States). Fuchsin + substrate chromogen (Agilent-Dako) or 3,39-diaminobenzidine (Kem-En-Tec Nordic A/S, Uppsala, Sweden) were used as chromogen. To visualize glucagon and PP, the ImmPRESS® HRP horse anti-rabbit IgG polymer detection kit (Vector Laboratories) was used.
Cellular composition of NPICCs and REPIs were quantified by QuPath software (version 0.3.2) using pictures scanned by uScope MXII slide scanner (Microscope International, Dallas, United States). The numbers of cells that stained positive for glucagon, somatostatin or PP were expressed as number of positive cells per 100 insulin positive beta cells. To assess vascularization, the area of CD31 positive cells (endothelial cell marker) were detected and normalized to the islet area.

After exsanguination, the mice were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, and the pancreas, thymus and bone were collected. Rabbit anti-insulin (Cell Signaling Technologies, Danvers, MA USA), rabbit anti-glucagon (Cell Signaling Technologies) and rabbit anti-TNF-α (Abcam, Cambridge, UK) were used as primary antibodies, followed by ImmPRESS reagent anti-rabbit IgG as the secondary antibody. The color was developed using an ImmPACT DAB kit (Vector Laboratories, Burlingame, CA, USA). For immunofluorescence analysis, the sections were incubated with anti-Vcam-1 (Cell Signaling Technologies) or rabbit anti-NG2 (Proteintech, USA) and sheep anti-von Willebrand Factor (Abcam) primary antibodies at 4 °C overnight, followed by Alexa555 anti-rabbit IgG as a secondary antibody (Thermo Fisher Scientific Inc. Waltham, MA, USA). To examine the localization of CD8a T cells in the thymus, frozen sections of fixed thymus were stained with rat anti-CD8a (Clone 53-6.7, Biolegend) as primary antibody and Alexa488 anti-rat IgG (Thermo Fisher Scientific) as secondary antibody at 4 °C overnight. These fluorescent immunostained sections were mounted with a DAPI-containing medium (Vector Laboratories) and observed under a fluorescence microscope.