To identify infusion sites for the Trkb.t1 virus, we stained for the HA tag. Sections were blocked in a solution containing 2% normal goat serum (NGS), 1% bovine serum albumin (BSA), and 0.03% Triton X-100 (Sigma) for 1 h at room temperature. Then, sections were incubated with a primary antibody solution containing anti-HA (1:250; Millipore Sigma), 2% NGS, 1% BSA, and 0.03% Triton X-100 at room temperature overnight. Sections were then incubated in a solution containing biotinylated secondary antibody (1:1000; Vector Laboratories), 1% NGS, and 0.03% Triton X-100 at room temperature for 1 h. HA signal was amplified by incubating sections in streptavidin Cy5 or Dylight 594 (15μ/mL; Vector Laboratories) for 30 min. Sections were mounted and coverslipped.
Cy5 streptavidin
Manufactured by Vector Laboratories
Sourced in United States
Cy5 Streptavidin is a fluorescently labeled streptavidin conjugate. Streptavidin is a tetrameric protein that binds to the vitamin biotin with high affinity. The Cy5 dye is attached to the streptavidin, providing a fluorescent label for detection and visualization applications.
Automatically generated - may contain errors
Lab products found in correlation
Dylight 594, by Vector Laboratories (2 mentions)
Anti ha, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Biotinylated secondary antibody, by Vector Laboratories (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti flag antibody, by Merck Group (2 mentions)
Maackia amurensis lectin 2 mal 2, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Anti ha magnetic beads, by Selleck Chemicals (1 mentions)
Anti gapdh mouse monoclonal antibody, by Proteintech (1 mentions)
5 protocols using cy5 streptavidin
1
Visualizing Trkb.t1 Virus Infusion Sites
2
Visualizing Trkb.t1 Virus Infusion Sites
3
Immunofluorescence Staining of MBL2 in A549 Cells
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A549 cells were cultured and fixed and blocked as previously described. Cells were stained with MBL2 (10 μg/mL in MBL2 binding buffer [20 mM Tris, 1 M NaCl, 10 mM CaCl2, 15 mM NaN3, 0.05% triton X-100, pH 7.4]; 1 h, 37 °C; Abcam). After washing with MBL2 wash buffer (10 mM Tris, 145 mM NaCl, 5 mM CaCl2, 0.05% tween-20, pH 7.4) 3×, cells were stained with mouse monoclonal anti-MBL (Biotin) (2 μg/mL in MBL2 wash buffer; 1 h, 37 °C; Abcam) and mouse monoclonal anti-influenza A that had been labeled with Cy3 dye and dialyzed following the manufacturers protocol (1 μg/mL in MBL2 wash buffer; 1 h, 37 °C; Abcam). Cells were washed 3× (MBL2 wash buffer) and stained with Cy5 Streptavidin (10 μg/mL; Vector Laboratories) in MBL2 wash buffer for 1 h at 37 °C (5% CO2). Cells were then washed 3× (MBL2 wash buffer) and stained with DAPI (600 nM in MBL2 wash buffer; 5 min, RT; ThermoFisher) before imaging. Samples were imaged by fluorescence microscopy as described above. Endo H-treated controls were prepared as described above.
The University of Georgia Institutional Animal Care and Use Committee approved all experiments under the Animal Use Protocol 2015-04-007, and the experiments were conducted in accordance with ref. 55 , The Animal Welfare Act, and ref. 56 .
The University of Georgia Institutional Animal Care and Use Committee approved all experiments under the Animal Use Protocol 2015-04-007, and the experiments were conducted in accordance with ref. 55 , The Animal Welfare Act, and ref. 56 .
4
Antibodies and Reagents for IAV Research
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The antibodies used in experiments were anti-GAPDH and anti-HA mouse monoclonal antibodies (catalog no. PMK043F and PMK013C; PMK Bio, Wuhan, China); anti-Flag mouse monoclonal antibodies (catalog no. F1804; Sigma, Saint Louis, MO, USA); anti-IAV NP, M1 and HA rabbit polyclonal antibodies (catalog no. GTX125989, GTX125928 and GTX127357; GeneTex, Irvine, CA, USA); anti-CMAS rabbit polyclonal antibody (catalog no. WG-04641; ABclonal, Wuhan, China); Alexa Fluor 594-conjugated AffiniPure goat anti-rabbit and Alexa Fluor 488-conjugated AffiniPure goat anti-mouse secondary antibodies (catalog no. GR200G-43C and GM200G-02C; Sungene Biotech, Tianjin, China). The reagents used in experiments were DAPI (4′,6-diamidino-2-phenylindole; 1:1000) (catalog no. C1002; Beyotime, Shanghai, China); Biotinylated Sambucus Nigra (SNA) and Maackia Amurensis Lectin II (MAL II) lectins (catalog no. B-1305-2 and B-1265-1; Vector Lab, Burlingame, CA, USA) and Cy5-Streptavidin (catalog no. SA-1500-1; Vector Lab, Burlingame, CA, USA). We also used Protein A/G Magnetic Beads (catalog no. HY-K0202; MCE, Shanghai, China) and anti-HA immunomagnetic beads (catalog no. B26202; Bimake, Houston, TX, USA).
5
Antibodies and Reagents for Protein Analysis
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The following antibodies and reagents were used in this study: anti-GAPDH mouse monoclonal antibody (Cat NO. 60004-1-Ig, Proteintech, Wuhan, China); anti-FLAG mouse monoclonal antibody (Cat NO. F1804, Sigma, Saint Louis, MO, USA); anti-HA mouse monoclonal antibody (Cat NO. M180-3, MBL, Beijing, China); anti-Lamin A/C rabbit polyclonal antibody (Cat NO. ET7110-12, HUABIO, Hangzhou, China); anti-TNPO3 rabbit monoclonal antibody (Cat NO. ET7108-80, HUABIO, Hangzhou, China); anti-IAV NP and M1 rabbit polyclonal antibodies (Cat NO. GTX125989 and GTX125928, GeneTex, Irvine, CA, USA); fluorescein labeled affinity purified antibody to mouse IgG (H+L) (Cat NO. 5230-0427, KPL, Milford, MA, USA) and Cy3-labeled antibody to Rabbit IgG (H+L) (Cat NO. 5230-0359, KPL, Milford, MA, USA); Horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies (BF03001 and BF03008, Beijing Biodragon Inmmunotechnologies, Beijing, China); DAPI (4′, 6-diamidino-2-phenylindole) (Cat NO. C1002, Beyotime, Shanghai, China); biotinylated Sambucus nigra (SNL) and Maackia amurensis Lectin II (MAL II) lectins (Cat NO. B-1305-2 and B-1265-1, Vector Lab, Burlingame, CA, USA); Cy5-streptavidin (Cat NO. SA-1500-1, Vector Lab, Burlingame, CA, USA); anti-Flag Magnetic beads (Cat NO. HY-K0207, MCE, Shanghai, China); and anti-HA Magnetic beads (Cat NO. B26202, Bimake, Shanghai, China).
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