Immunostaining of Duodenal Muscularis Externa

Immunostaining of duodenal muscularis externa was performed as previously described with minor modifications (65 (link), 66 (link)). Briefly, duodenal muscularis externa isolated from WT and IP6K2^−/− mice were blocked with 3% bovine serum albumin blocking solution containing the corresponding isotype control antibodies (IgG2a, R&D systems, Cat# MAB003; IgG2b, Dako) for 2 days after fixation with 4% paraformaldehyde overnight. The muscle layers were then washed with PBS containing 0.05% Triton X-100 and incubated with diluted primary antibodies against HuC/D (Thermo Fisher Scientific) or βIII-Tubulin (Biolegend, Cat# 801201, RRID: AB_2313773) for 3 days. After rinsing thrice with 0.05% Triton X-100 in PBS, the muscularis externa were then incubated with secondary goat anti-mouse IgG Alexa 594 (1:350, A-11020, Thermo Fisher Scientific) for 3 h at room temperature. The samples were then washed thrice with 0.05% Triton X-100 in PBS and mounted using antifading medium (12.5 mg/ml DABCO, 90% glycerol, pH 8.8 in PBS). Fluorescence images were obtained using an LSM 880 confocal microscope (Carl Zeiss).