Immunohistochemical Analysis of 5-HT and SP in Shrews

Shrews (n = 5–6 shrews per group) were treated with either vehicle or yohimbine (1 mg/kg, i.p.) and rapidly anesthetized with isoflurane and subjected to perfusion at 15 min and 30 min post-treatment to examine 5-HT and SP immunoreactivity. The experimental procedure prior to staining was performed as described above for Section 4.4.1. c-fos Staining and Image Analysis. Coronal brainstem sections (20 μm) were blocked with 0.1 M PBS containing 10% donkey serum and 0.3% Triton X-100, then incubated overnight at 4 °C with a mix of goat anti-5-HT primary antibody (1:1000, ab66047, Abcam) and rat anti-SP primary antibody (1:400, MAB356, EMD Millipore, Burlington, VT, USA) in 0.1 M PBS containing 5% donkey serum and 0.3% Triton X-100. Sections were washed 3 times (10 min each) in PBS and incubated in a mix of Alexa Fluor 488 donkey anti-goat (1:500, ab150133, Abcam) and cy3-conjugated donkey anti-rat (1:500, AP189C, EMD Millipore) secondary antibody in 0.1 M PBS containing 0.3% Triton X-100 for 2 h at room temperature. After washing with PBS 3 times (10 min each), sections were mounted with anti-fade mounting medium containing DAPI (Vector Laboratories). Images for the DVC were acquired using a confocal microscope (Zeiss LMS 880) as described above. Fluorescence intensity (mean gray value) of 5-HT and SP values were acquired using ImageJ software, as described previously [45 (link)].