Anti trfp

Mice were anesthetized with isoflurane and transcardially perfused with ice-cold PBS, followed by 4% (w/v) paraformaldehyde (PFA) in 0.1 M PB. Extracted brains were submerged in PFA for an hour before transferring to 30% (w/v) sucrose in 0.1 M PB for cryoprotection. After 48 h, brain were sectioned at 40 μm and stored in PBS-Azide. Sections were first permeabilized in 0.2% Triton-X100 in PBS (30 min, RT) and later incubated in (i) a blocking solution of in PBS containing 5% donkey serum, 3% bovine serum albumin and 0.2% Triton-X100 (1 h, RT); (ii) the same solution containing the primary antibody overnight at 4 °C (anti-tRFP, 1:1000, Evrogen; anti-GFP, 1:1000, Abcam; anti-GABA, 1:2000, MilliporeSigma; anti-parvalbumin, 1:2000, Abcam; anti-somatostatin, 1:1000, Peninsula Laboratories) (iii) the same solution containing a 1:500 dilution of secondary antibody (Alexa Fluor, ThermoFisher; 2 h, RT). All sections were then stained with DAPI (1:5000 diluted in 0.2% Triton X-100 in PBS, Sigma-Aldrich), washed in 0.2% Trixton-X100 in PBS and mounted with FluorSave (Merck Millipore). Images of the immunostained sections were acquired using the widefield (Nikon Instruments, Tokyo, Japan) or confocal microscope (Zeiss Microscopy, Munich, Germany).

Well-fed worms were washed three times with M9 buffer and then lysed using repeated freeze-thaw and sonication in a lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100 supplemented with protease and phosphatase inhibitor cocktails) and centrifuged at 130,000 g for 5 min. Protein quantification of the soluble sample was performed by a colorimetric detection reagent (BCA protein assay, Pierce). Equal amounts of protein were subjected to SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes for immunoblotting. After probing with primary antibody (anti-actin (I-19), Santa Cruz, Dallas, TX, USA, anti-CHCHD10 Proteintech; anti-tRFP Evrogen), the corresponding peroxidase-conjugated secondary antibody was detected by ECL western blot reagents (Pierce). ECL images were captured by the Fuji/GE LAS-4,000 imager (LAS-4,000, Pittsburgh, PA, USA) and quantified using ImageJ software (NIH ImageJ).

The following primary antibodies were used: Mouse monoclonal anti-FLAG [1:1,000 (IF), 1:20,000 (WB), Sigma], mouse monoclonal anti-Myc [1:1,000 (IF), 1:1,000 (WB), Cell Signaling], rabbit polyclonal anti-Actin (1:1,000, Sigma), rabbit polyclonal anti-tRFP (1:2,000, Evrogen), and rabbit polyclonal anti-IncA (1:200, kind gift from T. Hackstadt, Rocky Mountain Laboratories). The following secondary antibodies were used: Peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Jackson ImmunoResearch), peroxidase-conjugated goat anti-mouse IgG (1:10,000, Jackson ImmunoResearch), goat anti-mouse AlexaFluor 488 or 514 (IF: 1:1,000, Molecular Probes), and goat anti-rabbit Pacific Blue (IF: 1:1,000, Molecular Probes).