Immunohistochemical Analysis of c-Fos Expression in Brainstem and Jejunum after ZD7288-Induced Vomiting

To analyze c-Fos expression evoked at 90 min after ZD7288-induced vomiting, c-Fos immunostaining was conducted on both brainstem (20 µm) and jejunal sections (25 µm) from vehicle-treated and ZD7288 (1 mg/kg, i. p.)-treated animals (n = 4 shrews per group for brainstem staining; n = 3 for jejunual sections staining). Rabbit anti-c-Fos polyclonal antibody (1:5000, ab190289, Abcam) and Alexa Fluor 594 donkey anti-rabbit secondary antibody (1:500, Abcam) were used. The criterion used to differentiate the AP, NTS and DMNX within the DVC has been well recognized (Darmani et al., 2008 (link); Ray et al., 2009a (link); Chebolu et al., 2010 (link); Zhong et al., 2016 (link); Zhong et al., 2018 (link); Zhong et al., 2019 (link); Zhong and Darmani, 2020 (link)). For each animal, c-Fos positive cells in the AP, both sides of NTS and DMNX from 3 sections at 90-μm intervals were counted manually by an experimenter blind to experimental conditions. The average value was used in statistical analysis. Co-staining of jejunum sections with anti-c-Fos antibody and anti-NeuN (neuronal marker) antibody (1:300, MAB377, Millipore) followed by Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-mouse secondary antibodies (1:500, Invitrogen) was conducted to confirm neuronal localization of c-Fos.