Multicolor Immunofluorescent Tissue Labeling

Tissue sections were blocked for 15 min in True Black Supressor (Biotium, Fremont, CA, USA) followed by 15 min in Carbo Free Blocking Solution (Vector Laboratories, Newark, CA, USA) with 0.3% Tx-100. Sections were then incubated overnight at 4 °C in Carbo Free w/0.3% Tx-100 and rabbit anti-PDGFR-β (1:50; Thermo Fisher, Waltham, MA USA #MA5-15143), Alexa 488-Milli-Mark^® FluoroPan Neuronal Marker (1:100; Millipore Sigma #MAB2300X), Cy3-GFAP (1:100; Millipore Sigma, St. Louis, MO, USA #C9205), and DyLight 649-tomato lectin (1:1000; Vector Laboratories, Newark, CA, USA #DL-1178-1). Sections were then incubated in Carbo Free with 0.3% Tx-100 and Alexa 405 Plus goat anti-rabbit (1:200; Thermo Fisher # A48254) for 1 h at room temperature and coverslipped in ProLong Diamond (Thermo Fisher # P36970). When labeling Aquaporin-4 instead of GFAP, Alexa 488-Aquaporin-4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA # sc-32739 AF488) and the Milli-Mark Pan Neuronal Marker antibody was labeled with CF555 Mix-n-stain labeling kit (Biotium) according to the manufacturer’s instructions. A list of labeling reagents used in this study can be found in Table 1.