Goat anti guinea pig alexa 647

On day 12 of differentiation, iPSNs were plated in 24 well optical bottom plates (Cellvis). At day 32 of differentiation, iPSNs were fixed and immunostained as previously described [3 (link)]. Antibodies for immunostaining are as follows: 1:500 Rabbit Anti-Lamin B1 (Abcam ab16048), 1:1000 Guinea Pig Anti-Map2 (Synaptic Systems 188,004), 1:1000 Goat Anti-Rabbit Alexa 488 (Invitrogen A11034), 1:1000 Goat Anti-Guinea Pig Alexa 647 (Invitrogen A21450). iPSNs were imaged using a Zeiss LSM 800 confocal microscope. All images were acquired using identical imaging parameters (e.g. laser power, gain). Unless otherwise indicated, images presented are maximum intensity projections generated in Zeiss Zen Blue 2.3.

Sections containing the prelimbic cortex (Bregma: +2.46 ± 0.3 mm), the NAC (Bregma: +1.42 ± 0.2 mm), the posterior NAC, the dorsal striatum and the LS (Bregma: +1.00 ± 0.2 mm), the BNST (Bregma: -0.22 ± 0.3 mm), the hippocampus (Bregma: -1–70 ± 0.3 mm), or the amygdala (Bregma: -1.40 ± 0.2 mm) were incubated with primary antibodies diluted in the blocking solution: rat anti-5-HT (Millipore #MAB352, 1:100) for 48 h at room temperature followed by rabbit anti-SERT (Millipore #PC177L, 1:1000) and the KO-validated (Fasano et al., 2017 (link)) guinea-pig anti-VGLUT3 (Synaptic System #135204), at 1:500 dilution [as per supplier’s recommendations and other studies (Puighermanal et al., 2017 (link))], overnight at 4 degrees. After three washes in the blocking solution, the slices were incubated for 4 h at room temperature with secondary antibodies diluted in the blocking solution: goat anti-rabbit-Alexa 488, goat anti-guinea pig-Alexa 647 (Thermofisher Scientific, #A11034 and #A21450, 1:500) and goat anti-rat biotinylated (Jackson Laboratory # 112-065-003, 1:200). After three washes in the blocking solution, slices were incubated for 30 min in Streptavidin-Cy3 (Thermofisher Scientific #438315, 1:1000), washed three times in PBS, and mounted in Prolong Gold antifade mountant (Thermofisher Scientific, #P36934).

Fifty micrometers free-floating brain cryosections were used for immunohistochemistry or mounted for Nissl. For Nissl staining, mounted sections were dehydrated with ascending grades of alcohol and cleared with xylene using 0.1% Cresyl Violet (Sigma-Aldrich C5042) Acetate solution^{65 (link)}. Primary antibodies used were rabbit anti-GFP (Thermo Fisher Scientific, Invitrogen A11122), rabbit anti-GFAP (Abcam, ab7260), mouse monoclonal anti-NeuN (Chemicon MAB377 clone A60), guinea pig anti-VGLUT2 (Merk Millipore AB2251-I), mouse monoclonal anti-Satb2 (Abcam, ab51502), rat monoclonal anti-Ctip2 (Abcam, ab18465), rabbit polyclonal anti-Cux1 (Santa Cruz Biotechnology, sc-13024), and mouse monoclonal anti human Rorb (Perseus Proteomics, PP-N7927-00); and secondary antibodies used were goat anti-rabbit-Alexa 488 (Life Technologies, catalog #A-11034), goat anti-guinea pig-Alexa 647 (Thermo Fisher Scientific, catalog #A21450), goat anti-rat Alexa 594 (Thermo Fisher Scientific, catalog #A-11007), and goat anti-rabbit Alexa 594 (Thermo Fisher Scientific, catalog #A-11037). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (D9542 Sigma).