Anti guinea pig alexa 555

For histological labeling of astrocytes, 100 μm thick coronal brainstem slices were prepared from perfusion-fixed P40 C57ML/6J male mice (CLEA Japan, Shizuoka, Japan). The slices were permeabilized by incubation in GSA-BSA-T - PBS solution with 10% BSA (Bovine Serum Albumin, Sigma-Aldrich, Germany), 1% GSA (Goat serum, normal donor herd, Sigma-Aldrich, Germany), and 0.1% Triton (Sigma-Aldrich, Germany) for 2 h on a shaker (40 rpm) at room temperature (25°). For primary antibody application, 200 μl of a mix containing Anti-NeuN (1/200, Polyclonal Guinea pig antiserum 266-004, Synaptic Systems, Germany), Anti-MAP2 (1/200, Polyclonal Chicken antibody, ab5392, AbCam, Japan) and Anti-AlphaTubulin (1/100, Monoclonal mouse antibody, CP06, Sigma-Aldrich, Germany) diluted in BSA-GSA-T was deposed on top of the slices in individual wells and slices were incubated for 15 h. After washing the slices 4 times with BSA-GSA-T, the slices were incubated with 3 secondary antibodies for targeting the 3 primary antibodies coupled to a different fluorescent reporters (Anti-mouse-Alexa488, Anti-guinea pig-Alexa555, Anti-chicken-Alexa591, ThermoFisher Scientific, Minato, JP) for 4h, rinsed again 4 times in PBS and prepared for microscopy.