Ab22625

Ileum dissected from a mouse was embedded in OCT at −80 °C. Cuts of 10 μm cryosections were obtained on a microscope slide and put in room temperature for 30 min, followed by fixation in 4% PFA for 15 min. Blocking of tissue was conducted in blocking buffer containing 5% normal goat serum and 0.1% Triton x-100 in 1xPBS and incubated for 1 h at room temperature. As for the primary antibodies, rabbit anti-GLP-1 (ab22625, Abcam, Waltham, MA, USA) 1:100 dilution in blocking buffer was applied for 24 h at 4 °C. IgG Goat anti-Rabbit Alexa Fluor Plus 555 (2 mg/mL) 1:250 dilution was applied for 2 h as the secondary antibody at room temperature. The cells were counterstained with a nuclear stain VECTASHIELD DAPI mounting medium for 1 h at room temperature.

NT-expressing cell detection was performed using an anti-NT specific antibody (3488-7), kindly provided by Nicolai J. Wewer Albrechtsen and Jens J. Holst, from the University of Copenhagen. Commercial antibodies were used to identify neuroendocrine cell (anti-chromogranin-A antibody, ab17064, Abcam, Cambridge, UK), GLP-1 (ab22625, Abcam) and GIP (ab30679, Abcam)-producing cells.
Antigen retrieval was performed in the microwave using 10 mM citrate buffer (pH 6.0). Endogenous peroxidase was blocked with 3% H₂O₂ for 20 min, followed by incubation with normal serum for 30 min. Incubation with primary antibodies (Anti-chromogranin-A 1:200; Anti-NT 1:5000 Anti-GIP 1:500 and Anti- GLP-1 1:4000 in 5% BSA) was performed overnight at 4ºC. Incubation with secondary biotinylated polyclonal antibodies (1:200, EO35301-2 or EO35401-2, Dako, Glostrup, Denmark) was performed for 30 min, followed by the application of avidin–biotin complex (ABC) (1:100 dilution in 5% BSA; Vector Laboratories, Peterborough, UK) for 30 min. Diaminobenzidine was the chosen chromogen (3,3’-Diaminobenzidine, Dako), and the revelation lasted 2 min for chromogranin-A, 30 s for GIP and GLP-1 and 10 s for NT. All sections were counterstained with Mayer’s hematoxylin (HX390929, Merck, Darmstadt, Germany).