Ab150180

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.

NPY-1R was visualized on AF cells by immunofluorescent labeling using anti-NPY-1R antibodies. The rabbit AF cells were cultured in 8-well chamber slides (Nunc lab-Tek II, Chamber Slide System, ThermoFisher Scientific) with F-12, 10% FBS, 1% penicillin-streptomycin at 37°C, and 5% CO₂ until 50% confluence. The AF cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature. After washing in PBS, they were incubated with 10% goat serum, 1% BSA, and 0.1% Triton X-100 in PBS at room temperature for 30 minutes. Then AF cells were incubated with sheep anti-NPY-1R antibody (1:200, ab35336, Abcam plc) applied overnight at 4°C. Next, the cells were washed twice with 1x PBS and incubated with the donkey polyclonal secondary antibody to sheep IgG (1:500, Alexa Fluor 594, ab150180, Abcam plc) at a dilution of 1:500 for 60 minutes at room temperature. At last, they were counterstained with DAPI (4’,6-diamidino-2-phenylindole) at a dilution of 1:500. Fluorescence analysis was performed with a microscope (Eclipse TE2000-U Inverted Microscope, Nikon).
AF cells incubated with secondary, but no primary antibody was used as a negative control. Jurkat cells, an immortalized line of human T lymphocytes, were used as a control and incubated with the primary and secondary antibody as described above to establish antibody specificity.