Quantification of Enteroendocrine Cell Innervation

L-cells and EC cells were immunolabeled using antibodies against GLP-1 (1:200, SC-7782 Santa Cruz Biotechnology, Dallas, TX, USA) and 5-HT (1:400, ab66047 Abcam, Cambridge, UK), respectively. Neuronal innervation was assessed using pan-neuronal markers, protein gene product (PGP) 9.5 (1:800, 7863-0504 AbD Serotec), and the GLP-1 receptor (1:400, AGR-021 Alomone Labs). Staining for pERK (1:200, 4370 Cell Signalling, Danvers, MA, USA) and pCamKII (1:200, ab171095 Abcam) was performed following Ussing chamber experiments. Briefly, 10 μm sections were washed with blocking buffer. The primary antibody was applied (19 h, 4 °C), and tissues were washed with phosphate-buffered saline (PBS) and incubated (60 min, room temperature) with species-specific Alexa Fluor-conjugated secondary antibodies (1:200, Invitrogen, Carlsbad, CA, USA). A Leica DM4000 epifluorescence microscope was used to visualize immunoreactivity (IR) and images captured with a QImaging camera. Immunopositive cells for each group were manually counted in each section and averaged over five fields of view, as previously described [1 (link)]. Neuronal labeling was quantified using ImageJ software, where five fields of view (1.44 megapixel) were analyzed with ImageJ for the total number of immunoreactive pixels in the region of interest within the mucosal layer.