Rabbit anti ibai

Preparation of rabbit anti-GPHR and anti-mGluR1α antibodies were described previously (Maeda et al., 2008 (link); Kamikubo et al., 2013 (link)). Goat anti-calbindin [(Af1040), RRID: AB_2571569], guinea pig anti-calbindin [(Af-280), RRID: AB_2571570], anti-parvalbumin [(Af-1000), RRID: AB_2571615], rabbit anti-GABA_A receptor α1 subunit [GABA_A Rα1; (Af-660), RRID: AB_2571571], anti-vesicular GABA transporter [vGAT; (Af-500), RRID: AB_2571622], anti-type-1 vesicular glutamate transporter [vGlut1; (Af-280), RRID: AB_2571616], and anti-vGlut2 [(Af-720), RRID: AB_2571619] were purchased from the Frontier Institute. Mouse anti-NeuN [(A60), RRID: AB_2298772] was purchased from Millipore. Rat anti-Mac 2 [(M3/38), RRID: AB_10060357] was purchased from Cedarlane. Rabbit anti-IbaI [(019-19741), RRID: AB_839504] was purchased from Wako Pure Chemical Industries. Mouse anti-GM130 [(35/GM130), RRID: AB_398141] was purchased from BD Biosciences. Sheep anti-TGN38 [(AAHP499), RRID: AB_2287346] was purchased from AbD Serotec. A rat monoclonal antibody against GPHR was raised in rat using the peptide Cys-AHKQAPEKHMAP as the antigen. Rat hybridoma cells (Clone 35C5) were obtained by standard approaches.

Mice were deeply anesthetized with a mixture ketamine/xylazine and transcardially perfused with cold PBS, followed by cold 4%PFA. Brains were post-fixed overnight in 4% PFA at 4 °C. 30 μm serial coronal floating sections were obtained using a vibratome. For single immunofluorescence, sections were blocked with 10% donkey serum for 30 min at room temperature and then incubated with either mouse anti-NeuN (1:500, Millipore), mouse anti-GFAP (1:400, Abcam), rabbit anti-IbaI (1:500, WAKO), rabbit anti-Synaptotagmin 2 (Syt2) (1:400, Novusbio), rabbit anti-Otoferlin (Otof) (1:400, Arigo Laboratories), mouse anti-EAA1 (1:200, BD Transduction laboratories), rabbit anti-GABAR (1:400, Arigo Laboratories), overnight at 4 °C. The sections were washed with PBS before and after being incubated with Cy3-, Cy5- or Alexa-conjugated secondary antibodies (1/200, Jackson Immunoresearch) for 2 h at room temperature in blocking buffer. All sections were mounted onto gelatin-subbed slides and cover-slipped using Mowiol with DAPI. Images were obtained using a Zeiss Apotome2 fluorescence microscope and analyzed with ImageJ software.