Ab236654

Immunohistochemistry and histology were performed according to the procedure described in previous reports [12 (link)]. Briefly, the excised distal colons were fixed with 4% paraformaldehyde for 24 h at 4 °C. The frozen sections (10 μm) for histology were then routinely stained with hematoxylin and eosin (H&E). The frozen sections (30 μm) for immunohistochemistry were exposed to a rabbit anti-human GPR41 antibody (ab236654, Abcam, Cambridge, MA, USA), goat anti-rat CGRP antibody (ab36001, Abcam) or rat anti-mouse F4/80 (MCA497GA, BioRad, Hercules, CA, USA) for 12–18 h and then incubated with the appropriate secondary antibodies for 2 h (Alexa 488 donkey anti-rabbit IgG), Cy3 donkey anti-rabbit IgG, Cy3 donkey anti-goat IgG or Alexa 488 donkey anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The immunostained sections were examined using a confocal microscope (LSM700 & LSM780, Carl Zeiss, Oberkochen, Germany). To avoid selection bias, three representative preparations were selected from each mouse colon and then three mucosal sites of each preparation were quantitatively analyzed using open-source software ImageJ (ImageJ bundled with 64-bit Java 1.8.0_172; a Java-based image processing program developed at the NIH) [13 ].