Prolong gold antifade fluorescence media with dapi

Forty-micrometre-thick vibratome-cut brain sections from perfused rats were rinsed in PBS and blocked for at least 1 h in 3% normal goat serum/0.01% Tween 20. Sections were incubated in the following primary antibodies: mouse anti-regulator of G-protein signalling 14 (UC Davis/NIH NeuroMab Facility, AB_10698026, 1:1,000) or mouse anti-STEP (Cell Signaling, 4817, 1:500) and rabbit anti-PCP4 (SCBT, sc-74186, 1:500). Antibodies were diluted in blocking solution and sections were incubated for 24 h. After several rinses in PBS/Tween, sections were incubated in secondary antibodies (Alexa goat anti-mouse 488 and Alexa goat anti-rabbit 568, Invitrogen, 1:500) for 2 h. Finally, sections were washed in PBS/Tween and mounted under ProLong Gold Antifade fluorescence media with DAPI (Invitrogen).

Adult Amigo2-EGFP mice were anesthetized by isoflurane inhalation and transcardially perfused with 4% paraformaldehyde in PBS. Brains were postfixed for 24 h, submerged in 30% sucrose in PBS, and sectioned coronally at 40 µm on a cryostat. Sections were washed in PBS, blocked for at least 1 h in 5% normal goat serum (NGS, Vector Labs) diluted in 0.1% PBS-X (0.1% Triton X-100 in PBS) at room temperature, and incubated in primary antibodies diluted in the same buffer overnight. A chicken polyclonal anti-GFP antibody (Abcam) was used at a 1:2000 dilution to enhance Amigo2-EGFP fluorescence with either a rabbit polyclonal anti-PCP4 antibody (Santa Cruz) or a rabbit polyclonal anti-Wfs1 antibody (ProteinTech). Sections were thoroughly washed in 0.1% PBS-X and incubated in secondary antibodies (Alexa Fluor goat anti-chicken 488 and Alexa Fluor goat anti-rabbit 568, Invitrogen) diluted at 1:500 for 2 h at room temperature. Finally, sections were washed in 0.1% PBS-X and mounted under ProLong Gold Antifade fluorescence media with DAPI (Invitrogen). Sections were then imaged on a Zeiss 710 meta confocal microscope using a 40× oil-immersion lens.