Hindbrains were cryosectioned at 20 μm thickness and stained for selected antigens. After blocking in 10% normal horse serum in Tris-phosphate buffered saline (TPBS, pH 7.4) sections were incubated overnight in a primary antibody against ionized calcium binding adaptor molecule 1 (Iba1, 1:1000; 019-19741, Dako, GA) followed by an Alexa-488 secondary antibody (1:400; A21206, Invitrogen, CA) to visualize microglia activation as previously described (Gallaher et al. 2012 (link)). For visualization of vagal afferents, the hindbrain sections were incubated with isolectin B4 biotin-conjugated antibody (IB4, 1:400, cat# B-1205, Vector Laboratories, CA) for 12 h at room temperature (Shehab 2009 (link)), followed by ExtrAvidin-CY3 (1:600, E-4142, Sigma-Aldrich) for 2 h. Negative controls were performed by omission of primary antibodies. Sections were mounted in ProLong (Molecular Probes, OR) and examined under Nikon 80-I fluorescent microscope as previously described (Gallaher et al. 2012 (link), Peters et al. 2013 (link)).