Ab77192

Pancreases or isolated islets from Wistar rats, GK rats and human subjects were fixed in 4% formalin, paraffin embedded and cut into 5‐μm sections. For immunofluorescence, sections were dewaxed and rehydrated as described above. All samples were subjected to antigen retrieval by heating in 0.01 M sodium citrate solution before immunolabelling. Sections were incubated overnight using antibodies targeting PYY (ab22663), insulin (in house), glucagon (G‐2654, Sigma, Gillingham, UK), somatostatin (sc‐25262, Santa Cruz, Insight Biotechnologies, Wembley, UK), PP (ab77192), NPY1R (ab91262, Abcam, Cambridge, UK), NPY2R (ab31894, Abcam), NPY4R (HPA027863, Sigma), NPY5R (ab32886, Abcam) or DPP‐IV (ab28340, Abcam). The tyramide amplification system was used as a secondary antibody system to improve visualization of PYY in human samples, all NPYR proteins and DPP‐IV (ThermoFisher, Loughborough, UK). All other proteins were visualized using fluorescently‐ and HRP‐labelled secondary antibodies for immunolocalization as follows; anti‐rabbit 488 (A11034, Life Technologies, Loughborough, UK), anti‐mouse TRITC (F5262, Sigma), anti‐guinea pig 648 (ab150187, Abcam), anti‐rabbit HRP (P0448, Dako, Cheadle, UK). Images were visualized using a BioRad (Radiance 2100) confocal microscope.

IF staining was performed as previously described^{25 (link)}. The following primary antibodies were used: guinea pig anti-insulin and rabbit anti-insulin (ab7842 and ab63820; Abcam, Cambridge, UK), rabbit antiglucagon and sheep antiglucagon (ab92517 and ab36232; Abcam), rabbit anti-GLP1 and mouse anti-GLP1 (ab22625 and ab23472; Abcam), mouse anti-GLP1R (sc390773; Santa Cruz, Dallas, TX, USA), rabbit anti-GLP1R (NBP1-97308; Novus, CO, USA), mouse antisomatostatin (sc-74556; Santa Cruz), goat antipancreatic polypeptide (Ab77192; Abcam), rabbit anti-Ki67 (ARG53222; Arigo, Taiwan, China), rabbit anti-pancreatic and duodenal homeobox 1 (PDX1) (ab47267; Abcam), rabbit anti-NK6 homeobox transcription factor-related locus 1 (NKX6.1) (NBP1-49672SS; Novus), rabbit anti-forkhead box O1A (FoxO1A) (ab39670; Abcam), rabbit anti-octamer-binding transcrition factor-4 (OCT4) (GTX101497; GeneTex, CA, USA), rabbit anti-neurogenin3 (Ngn3) (2325032; Millipore, Boston, MA, USA), and mouse anti-Nestin (GTX630201; GeneTex). Detection was performed using secondary antibodies conjugated to Alexa488 (ab150185 and ab150077; Abcam), Alexa594 (ab150088, ab150132, ab150116 and ab150180; Abcam), or Alexa647 (ab150115; Abcam) fluorescent dye. 4'6'-diamidino-2-phenyl-indole (DAPI) (Invitrogen, Carlsbad, CA, USA) was used for nuclear staining.