Immunofluorescent Staining Protocol for c-Fos and NPSR

These sections were blocked with 10% bovine serum in PBS before they were incubated with a mixture of rabbit polyclonal antibody against c-Fos (1:1500) and goat anti-NPSR (1:500, sc-162893, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS containing 1% bovine serum for 72 h at 4°C on an agitator. After several rinses in PBS, the sections were incubated with Alexa Fluor^® 488-conjugated affinipure donkey anti-rabbit IgG (1:400, 711–545–152, Jackson ImmunoResearch Laboratories, Inc., PA, USA) and Cy™ 3-conjugated affinipure donkey anti-goat IgG (1:400, 705–165–147, Jackson ImmunoResearch Laboratories, Inc., PA, USA) for 24 h at 4°C. Primary antibody omission was used as a control. Finally, sections were mounted on slides, covered with a coverslip, using 50% glycerol in PBS, and observed under a fluorescence microscope and photographed under Zeiss LSM 710 laser confocal microscope. The specificity of the anti-NPSR antibody had been demonstrated in previous studies (Laitinen et al., 2004 (link); Shao et al., 2013 (link), 2016 (link)).