Ab28340

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.

Mouse neuroblastoma N2a cells (ATCC, CCL131) were grown in DMEM FCS (10%)-penicillin/streptomycin medium. Cells were plated in 6-well plates at a density of 100,000 cells/well. Five days after infection, cells were fixed for 20 min with paraformaldehyde (4%), washed three times with PBS, permeabilized for 5 min with Triton X-100 (0.1%), and blocked for 1 h with BSA (5%)/Tween (0.05%). The primary antibody against DPP4 (dilution of 1/500, Abcam ab28340) was added overnight, and then, cells were rinsed three times with PBS and incubated for 1 h with the secondary anti-goat antibody (Interchim, dilution of 1/500 + DAPI (1/20,000)). Cells were then rinsed again with PBS 1× and fixed with the VectaMount medium (Vector Laboratories) before Cytation imaging analysis with the Biotek Cytation 5 microscope, which automatically acquired six pictures per well with the same acquisition parameters (×20 magnification, numeric aperture 0.45, brightfield, at following excitation and emission wavelengths, respectively: DAPI (377; 447); GFP (469; 525); Texas Red (586; 647)). We used ImageJ software to quantify the fluorescence intensity Texas Red in GFP-positive cells.

Total proteins of cells were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After being blocked with 5% non-fat dry milk in PBS, the membrane was incubated with antibodies to FAPα (1:1,000, AF3715; R&D Systems, Inc., Minneapolis, MN, USA), dipeptidyl peptidase 4 (DPP4; 1:1,000, ab28340; Abcam, Cambridge, MA, USA) or matrix metalloproteinase (MMP)2 (1:1,000, ab86607; Abcam) at 4°C overnight. After being washed several times, the polyvinylidene difluoride membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 hours. The bands were then detected by Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) according to the manufacturer’s protocols. β-Tubulin protein levels were also determined by using the specific antibody (1:3,000, ab126165; Abcam) as a loading control.