The synaptosomes were attached to the polylysine-coated coverslips for 40 min at 4 °C, fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min, permeabilized with 0.2% Triton X-100 for 60 min and incubated for 24 h with a mixture primary antibodies against vesicular transporter of glutamate type 1 (VGLUT1, 1:200; Abcam, Cambridge, UK) and GABAA receptorα1 subunit(1:100; Abcam, Cambridge, UK) for 90 min at room temperature. After rinsing with blocking buffer, the synaptosomes were incubated with a mixture of goat anti-mouse DyLight 549-and goat anti-rabbit fluoresceinisothiocyanate (FITC)-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. The synaptosomes were then washed three times with phosphate buffer and 0.1 M carbonate buffer (pH 9.2), and the coverslips were mounted with fluoromount (DAKO North America, Inc., Carpinteria, CA, USA). Images were acquired in an Image Xpress micro confocal (Molecular Devices, San Jose, CA, USA).
Dylight 549
Manufactured by Thermo Fisher Scientific
Sourced in United States
DyLight 549 is a fluorescent dye used for labeling biomolecules such as proteins, peptides, and nucleic acids. It has an excitation maximum at 562 nm and an emission maximum at 576 nm, making it suitable for detection in the orange-red region of the visible spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Dylight 488, by Thermo Fisher Scientific (2 mentions)
Fluoromount, by Agilent Technologies (1 mentions)
Imagexpress micro confocal, by Molecular Devices (1 mentions)
Goat anti rabbit fluorescein isothiocyanate fitc conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Vglut1, by Abcam (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Mouse anti caspase 1, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti asc, by Santa Cruz Biotechnology (1 mentions)
Goat anti nlrp3, by Abcam (1 mentions)
Fv500, by Olympus (1 mentions)
Hoechst 3342, by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
Mccoy s 5a medium, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
8 protocols using dylight 549
1
Synaptosomal Immunofluorescence Labeling
2
Biochemical Analysis of Signaling Pathways
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The materials and reagents were purchased from the companies indicated in parentheses: Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL-Life Technologies, NY, USA); fetal bovine serum (FBS, Thermo Scientific, Utah, USA); horse serum (Gibco-BRL-Life Technologies, NY, USA); protein G-agarose and protein A-agarose, antibodies against hnRNP M, p70 S6 kinase, SGK1 S422, PKCα S657, PKCζ and FoxO1 S319 (Santa Cruz Biotechnology, CA, USA); antibodies against mTOR, Rictor, Raptor, SGK1, PKCα, Akt1 S473, pan Akt1 and anti-GFP antibodies (Epitomics, CA, USA); antibodies against p70 S6 kinase T389, Akt T308, PKCζ T410 and FoxO1/FoxO3 T24/T32 (Cell Signaling Technology, MA, USA); antibodies against PKCζ T560, FoxO1, FoxO3, FoxO1 S256 and FoxO3 S253 (Abcam, Cambridge, UK); antibodies against FLAG and tropomyosin (Sigma, MO, USA); horseradish peroxidase anti-rabbit/anti-mouse secondary antibodies (Jackson, PA, USA); anti-rabbit/anti-mouse immunoglobulin G (IgG) antibodies conjugated to Alexa fluor 488 and anti-rabbit/anti-mouse immunoglobulin G (IgG) antibodies conjugated to DyLight 549 (Invitrogen, CA, USA); and Fluoromount G (Southern Biotech, AL, USA). The anti-FoxO3 S315 antibodies were a generous gift from Dr. Michael Greenberg (Harvard Medical School, Boston). All other chemicals were from Sigma (MO, USA) unless indicated otherwise.
3
Immunofluorescence Analysis of NLRP3, ASC, and Caspase-1
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Cells were grown on eight-well chamber slides and then treated as indicated group and then fixed in 4% paraformaldehyde for 15 min. The cells were washed in PBS and incubated overnight at 4°C with incubated using goat anti-Nlrp3 (1:100, Abcam), rabbit anti-ASC (1:50, Santa Cruz Biotech), or mouse anti-caspase-1(1:50, Santa Cruz Biotech) antibodies. Double immunofluorescence staining was performed by incubating slides with Dylight 488- or Dylight 549- labeled secondary antibody (1:800, Invitrogen) for another 1 h at room temperature. The slides were visualized through a confocal laser scan microscope (FV 500; Olympus Corporation, Tokyo, Japan). Colocalization in cells was analyzed by Image Pro Plus software, and the colocalization coefficient was represented by Pearson’s correlation coefficient (PCC).
4
Cell Line Cytotoxicity Assay Protocol
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All the cell lines were obtained from ATTC, USA. McCoy's 5a medium, MEM, RPMI-1640, FBS, BSA, isopropanol, dimethylsulfoxide (DMSO), formaldehyde, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Triton X-100, and PBS were obtained from Sigma, USA. Hoechst 3342 and Dylight 549 were obtained from Thermo Fisher Scientific, USA. All the antibodies, namely, BCL-2 (SC-738), p53 (SC-126), and cleaved caspase 3 (SC-22171), were procured from Santa Cruz Biotechnology, Inc., USA.
5
Immunofluorescence Staining of Blastocysts
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Blastocysts were fixed in 4% paraformaldehyde for 40 min at 4 °C, washed with PBS, permeabilized with 0.5% Triton X-100 in PBS for 20 min, and washed with PBS. The blastocysts were then blocked with 2.5% goat serum for 45 min, incubated overnight with primary antibodies at 4 °C, washed with PBS, incubated with the secondary antibodies for 30 min at room temperature, and washed with PBS. The combinations of primary and secondary antibodies are as follows. Anti-OCT4 (1:200 dilution; PM048, MBL Life Science, Nagoya, Japan) with goat anti-rabbit DyLight 549 (1:250 dilution, Thermo Fisher Scientific); and anti-CDX2 (1:150 dilution; B-MU392AUC, BioGenex, California, USA) with goat anti-mouse DyLight 488 (1:250 dilution, Thermo Fisher Scientific). Finally, blastocysts were whole-mounted with Antifade Mounting Medium with DAPI (H1200, Vector Laboratories) and observed under a confocal laser scanning microscope (C2+, Nikon, Japan). Z-stacks were collected at 0.4 μm intervals, and the maximum intensity projection images were generated from the z-stacks with total 10–12 μm thickness.
6
Monoclonal Antibody Generation Against K. pneumoniae CPS
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MAbs to ST258 CPS were generated by immunizing 6- to 8-week-old BALB/c mice with 100 μg of PA-conjugated CPS of K. pneumoniae strains 36 and 34 in complete Freund’s adjuvant, followed by boosters of PA-conjugated CPS in incomplete Freund’s adjuvant 2, 4, and 6 weeks later. Fusion and cloning were performed as previously described (8 (link)). The 8F12 and 17H12 variable regions were sequenced by GenScript and analyzed with the International ImMunoGeneTics Information System software program. The affinities of the MAbs were calculated by ELISA as previously described (8 (link)). Labelling of MAbs with DyLight 549 (ThermoFisher Scientific) was done following manufacturer’s protocol. Reduction of the multimeric form of IgM 1C9 MAb was done with 0.15 M of β-mercaptoethanol in TBS (1:1 v/v) for 1 h at 37°C as previously reported (44 (link)). Either commercial murine IgG3 (Crown Biosciences Inc.), which recognizes hen egg lysozyme, or 9D8, an IgG3 MAb that binds to arabinomannan (36 (link)), was used as an IgG control antibody.
7
Immunofluorescence Imaging of Cytokines
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Cells were tiled in a 24-well plate containing sterile cover glass slides and were cultured for 48 h, fixed with paraformaldehyde for 30 min, incubated with 0.3% Triton X-100 for 10 min, blocked with 3% goat serum for 1 h, and then incubated overnight with anti-Mcp-1 (1:50, Abcam, Eugene, USA), anti-Tnf-α (1:50, BBI Life, Shanghai, China), and anti-Gal-3 (1:50, Santa Cruz Biotechnology, USA). Then, cells were washed three times with PBS; DyLight488 or DyLight549 (Thermo Fisher Scientific, MA, USA) was then added into the cells for 1 h under dark conditions. Next, cells were washed with PBS three times and treated with DAPI for 10 min. Finally, the images were observed with a confocal microscope and were analyzed with LAS AF Lite (Leica, Solms, Germany).
8
Immunohistochemical Localization of Arabinogalactan Proteins
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Flower buds and flowers were prepared as described by Krawczyk et al. (2016) (link). After dehydration, material was embedded in Steedman’s wax and sectioned at 2–5 µm. Microtubules were visualized using a rat primary antibody against α-tubulin (Ab6161; Abcam, UK) and an anti-rat secondary antibody conjugated with DyLight™ 549 (AS12 2084; Thermo Fisher Scientific). The chromatin of the nuclei was stained with 7 µg ml−l DAPI (Sigma-Aldrich).
Rat monoclonal antibody JIM13 (Plant Probes, UK) was used to recognize arabinogalactan proteins (here, detection of the trisaccharide β-d -glucose A-β (1→3)-d -galactose A-α (1→2)-l -rhamnose; Knox et al., 1991 (link); Yates et al., 1996 (link)). Ten randomly selected small flower buds of both lines were sectioned to 2–5 µm and incubated with JIM13 (dilution 1/20 in PBS). After washes in PBS, the sections were incubated with anti-rat secondary antibody conjugated to FITC (Ab6840, Abcam; dilution 1/500 in PBS). In negative control experiments, the primary or secondary antibodies were omitted. The sections were mounted under coverslips with Mowiol medium and viewed under an epifluorescence microscope.
Rat monoclonal antibody JIM13 (Plant Probes, UK) was used to recognize arabinogalactan proteins (here, detection of the trisaccharide β-
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