Immunofluorescence Staining of Cortical Neurons

Immunofluorescence staining was performed as described previously (Fanibunda et al., 2019 (link)). In brief, cortical neurons were fixed in 4% paraformaldehyde, followed by blocking in 10% horse serum and incubated with primary antibodies, rabbit anti-pCREB (1:1000; Cell Signaling Technology, MA, United States) or goat anti-5-HT_2A receptor (1:500, Santa Cruz Biotechnologies, United States) along with the pan-neuronal marker, mouse anti-MAP2 (1:1000, Sigma-Aldrich, United States) overnight at 4°C. This was followed by incubation with secondary antibodies, Alexa 488 conjugated anti-goat (1:500; Molecular probes, CA, United States) or Alexa 488 conjugated anti-rabbit (1:500; Molecular Probes, CA, United States) or biotinylated horse anti-mouse (1:500, Roche Applied Science, Switzerland) with subsequent incubation with streptavidin-conjugated Alexa 568 (1:500, Molecular Probes, CA, United States) for 2 h. Following secondary antibody incubation and serial washes, cortical neurons were mounted in Vectashield (Vector Laboratories, CA, United States), and images were captured on the Zeiss LSM5 Exciter laser scanning microscope.