Immunofluorescence Analysis of Microglia Activation and Vagal Afferents

Standard immunofluorescence was used to determine microglia activation and vagal afferent density in the hindbrain. Sections were incubated overnight with a primary antibody against ionized calcium-binding adaptor molecule 1 (Iba-1, Wako, Richmond, VA, USA; Cat#019-19741, RRDI: AB_839504) followed by Alexa-488 secondary antibody to visualize microglia activation as previously described [5 (link)]. In addition, hindbrain sections were incubated with GSL I-isolectin B4 biotin-conjugated (IB4, Vector Laboratories Cat#B-1205, RRDI: AB_2314661) overnight followed by ExtrAvidin-CY3 (Sigma-Aldrich, St. Louis, MO, USA; Cat#E-4142) for 2 h to visualize primary unmyelinated vagal afferents innervating the GI tract as previously described [8 (link)]. Sections were mounted in ProLong (Molecular Probes, OR) and examined under a Nikon 80-I fluorescent microscope. The area fraction of Iba1 was analyzed using Nikon Elements AR software as previously described [18 (link),23 (link)].