Ab30637

The retinas of 0-24 hr old adult flies were dissected in phosphate buffer (pH 7.4). The cornea and the rest of the head including the brain tissue were removed using forceps. Subsequently, the retinas were fixed in 4% formaldehyde in phosphate buffer (pH 7.4) for 15 min, followed by a 30 min wash in phosphate buffer (pH 7.4). Prior to antibody staining, the retinas were kept in a 0.3% Triton X-100 phosphate buffer solution at 4° for 24 hr or longer. The antibody staining was done according to a standard protocol. The following antibodies were used: rat anti-Crb (F3, 1:500; Pellikka et al., 2002 (link)); mouse anti-Rh1 (4C5, 1:50; Developmental Studies Hybridoma Bank), guinea pig anti-Eys (G5, 1:500; Husain et al., 2006 (link)), mouse anti-Nervana (nrv5f7, 1:50, Developmental Studies Hybridoma Bank), rabbit anti-GM130 (ab30637, 1:300, Abcam). Secondary antibodies anti-guinea pig alexa fluor 647 (A21450), anti-rat alexa fluor 555 (A21434), and anti-mouse alexa fluor 488 (A11029) were used at 1:400 (Molecular Probes/Thermo Fisher Scientific). Acti-stain555 (PHDG1-A, 1:75, Cytoskeleton Inc) was used to visualize rhabdomeres. A Leica TCS SP8 confocal microscope with 100x oil objective (NA 1.4) was used to capture images. Image J (Fiji) and Adobe Photoshop and Adobe Illustrator were used to edit and compile figures.

Primary antibodies used for western blotting are listed below: Tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), ß-Actin (mouse, 1:2000, Santa Cruz), Rh1 (mouse, 1:2000, Developmental Studies Hybridoma Bank), TRPL (rabbit, 1:500, Fisher, Waltham, USA) [30 (link)], TRP (rabbit, 1:2000) [29 (link)], INAD(rat, 1:2000) [29 (link)]. The TRP and INAD antibodies were a gift form Dr. C. Montell. Secondary antibodies were bought from LI-COR Biosciences, which included IRDye 680 goat anti-mouse-IgG, IRDye 800 goat anti-rabbit-IgG, and IRDye 800 goat anti-rat-IgG antibodies (1:10000).
Primary antibodies used for staining are listed below: Rh1 (mouse, 1:200, Developmental Studies Hybridoma Bank), Cnx99A (mouse, 1:20, Developmental Studies Hybridoma Bank), GM130 (rabbit, 1:200, #ab30637, Abcam) GFP (rabbit, 1:200, Invitrogen), RFP (rat, 1:200, ChromoTek), EMC3 (rabbit, #ab175537, Abcam), NaK ATPase (mouse, #NA163540, Thermo Scientific), M-Opsin opsin (AB5405, Millipore, MA, USA), Rho 1D4 monoclonal antibody against rhodopsin was a gift from Dr. Robert Molday, University of British Columbia, Canada. Secondary antibodies were bought from Invitrogen (anti mouse, rabbit or rat IgG labeled with Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, or Alexa Fluor 488), 1:500 dilution.

CRISPR-mutations third-instar larvae raised at 25°C were harvested, dissected in PBS 1×, fixed in 4% paraformaldehyde for 15 min and washed twice in PBS containing 0.3% Triton X-100 (Sigma-Aldrich). Dissected larvae were probed with anti-GM130 (Abcam, ab30637, rabbit, 1:1000) overnight at 4°C, then washed 3 times with PBS plus 0.3% Triton X-100 and incubated with Alexa Fluor 555 anti-rabbit antibody (Molecular Probes Invitrogen, 1:500). Larvae were washed 3 times with PBS and mounted on coverslips using Mowiol (Sigma-Aldrich).
HeLa cells transfected as indicated above were fixed in PBS containing 4% paraformaldehyde for 15 min, incubated with 50 mM NH₄Cl for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 3 min and then blocked with 2% BSA and 0.2% gelatin for 30 min. Antibodies used were anti-GM130 (BD, #610822, mouse, 1:1000); anti-TGN46 (Abcam, ab50595, rabbit, 1:200). Alexa Fluor 555 conjugated goat anti-mouse and Alexa Fluor 647 conjugated goat anti-rabbit (Molecular Probes Invitrogen) were applied for 1 h at room temperature. Coverslips were mounted using Mowiol (Sigma-Aldrich).
All confocal images were acquired using a Leica TCS SP5 II confocal microscope, equipped with a PlanApo 100×/1.4 Oil objective, using a 543 nm laser line. Confocal microscopy imaging was performed at 1024 × 1024 pixels per image, with a 200 Hz acquisition rate.

Total proteins from cells or exosomes were extracted at 4 °C for 30 min by using RIPA Lysis Buffer (Beyotime, China). Adipose tissue samples were homogenized using a Teflon/glass homogenizer in lysis buffer with pH 7.4, containing 50 mM Tris-HCL buffer, 250 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 50 mM NaF, 1 mM orthovanadate, and protease inhibitors. Then, the lysates were centrifuged at 500g for 10 min at 4 °C and the supernatant was collected. Western blotting was performed according to the concentration of proteins determined by BCA method. Primary antibodies used in this study included anti-GM130 (1:1000, Abcam, ab30637), anti-CD63 (1:1000, Abcam, ab134045), anti-TSG101 (1:500, Santa, sc-7964), anti-CD9 (1:1000, Abcam, ab236630), anti-GAPDH (Proteintech, 60004-1-1 g) and anti-BMP7 (1:1000, Abcam, ab129156). Secondary antibodies were HRP-conjugated goat anti-mouse IgG (D110087, BBI, China) and HRP-conjugated goat anti-rabbit IgG (1:5000, Cell Signaling Technology, 7074P2).

For all images shown, cells were fixed for 20 min in 4% formaldehyde/PBS and permeabilized in 0.5% Triton X-100/PBS. For Table 2, cells were also fixed in methanol (5 min) and acetone (45 s), or as above but with 0.1% glutaraldehyde/2% paraformaldehyde/PBS and after permeabilization quenched with 1 mg/ml NaBH₄ in PBS for 5 min. Cells were blocked for 30-60 min in PBS containing 20% FCS and 0.25% Tween-20 and probed with the antibodies in the same buffer. Rabbit antibodies against Arl8 (Hofmann and Munro, 2006 (link)), GM130 (Abcam, ab30637, 1:500 dilution), Golgin245 (Sinka et al., 2008 (link)), Sec16 (Ivan et al., 2008 (link)), and the mouse and goat (1:2000) antibodies generated in this study were detected with species-specific Alexa-labeled secondary antibodies (Molecular Probes, 1:400), or IgG subclass-specific secondary antibodies (Invitrogen). For standard imaging, cells were mounted in VECTASHIELD (Vector Laboratories) and micrographs obtained with a confocal microscope (Zeiss LSM 780) or a wide-field microscope with Micro-Manager software [Zeiss Axioplan and CoolSNAP HQ2 (Photometrics)]. For STED microscopy, coverslips were mounted in Prolong Gold anti-fade mounting media (Invitrogen). Super resolution STED images were acquired with a LEICA TCS SP8 X microscope equipped with 592 nm and 660 nm depletion lines.