Cy3 conjugated goat anti mouse secondary antibody

Cells were grown on coverslips for at least 24 h. For internalization studies, they were incubated with or without 1 µM somatostatin-14 (Bachem, Bubendorf, Switzerland) or octreotide (peptides & elephants, Hennigsdorf, Germany) in serum-free medium for 30 min at 37°C. Cells were washed with PBS, fixated in 1:1 acetone/methanol for 2 min, and incubated with anti-SSTR2 primary antibody (sc365502, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:50 in PBS overnight at 4°C. After washing with PBS, cells were incubated with a Cy3-conjugated goat anti-mouse secondary antibody (#115-165-146, Jackson ImmunoResearch, West Grove, PA, USA) diluted 1:1,000 in PBS for 1 h at room temperature. After washing with PBS, coverslips were briefly dipped into 96% ethanol, air-dried, and mounted on glass slides with Immu-Mount (Thermo Fisher Scientific, Waltham, MA, USA). Mounted cells were imaged using a confocal laser scanning microscope (LSM510, Carl Zeiss, Jena, Germany) using a helium–neon laser at 543 nm, LP560 emission filter, and 40× or 63× NeoFluar oil immersion objectives. RIN-1038-SSTR2-GFP cells were not immunostained after fixation, but otherwise treated the same. To visualize GFP fluorescence, an argon laser at 488 nm, LP505 emission filter was utilized on the same microscope.

Marc-145 cells grown in 96-well plates were infected with the recombinant or parental virus preparations (0.01 MOI) for 36 h. After fixation using 4 % paraformaldehyde, cells were washed with PBS and incubated with 6D10, a monoclonal antibody to PRRSV N protein (produced in our lab), or a goat anti-porcine IL-4 polyclonal Ab (R&D Systems, MN) for 1 h. Cells were then washed with PBS and incubated for 1 h with a Cy3-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Inc, USA) or an Alexa Fluor 488-labeled donkey anti-goat secondary antibody (Abcam, UK), respectively. Images were taken using a Leica confocal microscope.

DDM (Affymetrix Inc., Maumee, OH, USA) was used to solubilize bovine opsin from ROS disc membranes. The β-Gal fragment complementation assay was performed with the Gal-Screen System (Applied Biosystem, Bedford, MA, USA). PNGaseF was purchased from NEB (Ipswich, MA, USA) for the deglycosylation of cell lysates. 4′,6′-Diamidino-2-phenyl-indole (DAPI) and Hoechst33342 were purchased from ThermoFisherScientific (Grand Island, NY, USA) for nuclear staining. Cy3-conjugated goat anti-mouse secondary antibody was ordered from Jackson ImmunoResearch Laboratories, Inc. (catalogue number: 115-165-146, West Grove, PA, USA) for immunostaining. DMSO and 9-cis-retinal were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Mouse monoclonal B6-30 and 1D4 anti-rhodopsin antibodies were purified from hybridoma cells^{60 (link)–62 (link)}. Alexa 488-conjugated B6-30 anti-rhodopsin antibody was obtained using the Alexa Fluor 488 Antibody Labeling Kit (ThermoFisherScientific). Forskolin was purchased from Tocris Biosciences (Bristol, UK). Scriptaid was purchased from Selleck Chemicals (Houston, TX, USA). YC-001 and its related compounds were synthesized and purified as described below under Medicinal Chemistry.

Labeling of GRNs expressing Gr5a and Gr61a was carried out by expressing a variant of the green fluorescent protein mCD8::GFP by means of the Gal4/UAS system. Adult flies aged over 10 days after eclosion were used, as these flies had accumulated a sufficient amount of mCD8::GFP. Reporter expression was detected either by direct observation of raw fluorescence under a fluorescence microscope (Microphot-FX; Nikon, Tokyo) or by immunostaining GFP with a rabbit anti-GFP antibody (Molecular Probes, Eugene, OR) in conjunction with a goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes), which was followed by observation under a confocal microscope (BioRad, Hercules, CA). Neuropiles in the CNS were labeled with the mouse monoclonal antibody nc82 (Developmental Studies Hybridoma Bank at the University of Iowa) and a Cy3-conjugated goat anti-mouse secondary antibody (Jackson Immuno Research, West Grove, PA).

To detect the double-strand breaks, the wing imaginal discs of the third instar larvae were dissected in ice-cold PBS and fixed for 1 h at room temperature in PBS containing 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). After washing and blocking for 1 h with PBS containing 0.1% Triton and 2% BSA, the samples were incubated at 4 °C overnight with anti-phospho-histone H2AX (γH2AX, Merck Millipore, Burlington, MA, USA). Subsequently, the samples were washed and incubated with the Cy3-conjugated goat anti-mouse secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA) for 2 h at room temperature. For visualization, the samples were mounted in VECTASHIELD mounting media (Vector Lab., Burlingame, CA, USA), and fluorescence images were acquired using a confocal microscope (LSM510 META, Carl Zeiss, Oberkochen, Germany). The ratio of γH2AX foci to nucleus area was quantified using ImageJ software. Data are presented as mean ± SEM values.

Each construct in the mutation library was tested for full-length TAS2R16 translation and surface expression by immunofluorescent antibody binding assays. The mutation library and controls were expressed in HEK-293T cells in 384-well plates as described^{21 (link)}. Twenty-four hours post-transfection, cells were washed with PBS-/- (HyClone), followed by addition of cell stripper (Cellgro). Suspended cells were fixed with paraformaldehyde at a final concentration of 4%. To detect surface expression of proteins, cells were incubated for one hour with anti-FLAG MAb M2 (1:500; Stratagene). To determine total (full-length) expression, cells were permeabilized using PBS++ (HyClone) with 0.1% saponin and incubated with an anti-V5 antibody (Invitrogen R960-25) for one hour. Primary antibody incubations were followed by a one hour incubation with goat anti-mouse Cy3-conjugated secondary antibody (1:500; Jackson Laboratories). Secondary antibody fluorescence was measured from a minimum of 500 cells by flow cytometry on an Intellicyt HTFC screening system. FLAG and V5 reactivities for library clones were normalized to the values obtained from wild-type TAS2R16.

SH‐SY cells grown on coverslips coated with Poly‐L lysine were fixed with 4%PFA (Electron Microscopy Sciences) for 15 min and then washed in D‐PBS. Cells were permeabilized with D‐PBS/0.2% Triton X‐100 for 1 h, then blocked in 3% Normal Donkey serum in D‐PBS/0.2% Triton X‐100 for 1 h. Next, cells were incubated O.N. at 4c with 1:100 dilution of anti‐NCX1 antibody (Abcam—ab2869) in blocking solution. Cells were washed in D‐PBS/0.2% Triton X‐100 and incubated for 1 h at RT with 1:500 dilution of goat anti‐mouse Cy3‐conjugated secondary antibody (Jackson immunoresearch 115‐166‐072). Finally, cells were washed in D‐PBS, stained with DAPI for 10 min, washed, and mounted on microscope slides.