The binding preference of A/SW1/18 for avian- and human-specific virus receptors was confirmed using a solid-phase direct binding assay, as described previously (Koo et al., 2018 (link)). In brief, 10 μg of fetuin (Sigma-Aldrich, MO, USA) was coated and incubated overnight at 4°C and blocked with 5% bovine serum albumin (BSA) in PBS at room temperature for 1 h. After blocking, the plates were washed four times with PBS and incubated with 64 hemagglutinating units (HAU) of each virus at 4°C overnight. After virus removal, the plate was washed as described above and incubated with 0.1 ml of each biotinylated glycan per well at different concentrations at 4°C for 3 h. Glycan binding was detected by adding horseradish peroxidase-conjugated streptavidin (Invitrogen, Carlsbad, CA, USA) followed by 3,3,5,5-tetramethylbenzidine substrate (Sigma-Aldrich), and the optical density was measured at 450 nm using a Synergy HTX multi-mode microplate reader (BioTek, VT, USA). A low-pathogenic H5N2 avian influenza virus, A/Aquatic Bird/Korea/CN2/2009 (A/CN2/09), with a binding preference for the avian receptor, was used as a control (Koo et al., 2018 (link)).
Horseradish peroxidase conjugated streptavidin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Horseradish peroxidase-conjugated streptavidin is a protein complex that consists of the protein streptavidin covalently linked to the enzyme horseradish peroxidase. This complex can bind to biotin-labeled molecules and catalyze a colorimetric or chemiluminescent reaction when a suitable substrate is present.
Automatically generated - may contain errors
Lab products found in correlation
Fetuin, by Merck Group (3 mentions)
3 3 5 5 tetramethylbenzidine substrate, by Merck Group (3 mentions)
Liquid dab substrate pack, by Thermo Fisher Scientific (3 mentions)
Tmb substrate, by Thermo Fisher Scientific (3 mentions)
Victor3 1420 multilabel counter plate reader, by PerkinElmer (2 mentions)
O phenylenediamine dihydrochloride, by Merck Group (2 mentions)
Subcellular protein fractionation kit, by Thermo Fisher Scientific (2 mentions)
Pentylamine biotin, by Thermo Fisher Scientific (2 mentions)
1 step ultra tmb elisa substrate solution, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Tween 20, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Corning (2 mentions)
Infinite m200 microplate reader, by Tecan (2 mentions)
Varioskan flash, by Thermo Fisher Scientific (2 mentions)
Biotinylated goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
3 amino 9 ethylcarbazole, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
61 protocols using horseradish peroxidase conjugated streptavidin
1
Glycan Binding Preference of Avian and Human Influenza Viruses
2
Immunohistochemical Analysis of β-Catenin and Cyclin-D1
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4 μm formalin-fixed, paraffin embedded slides were incubated with the rabbit anti human β-catenin antibody or rabbit anti human cyclin-D1 antibody (Sigma) after over night incubation at 4°C. The samples were then labeled with horseradish peroxidase conjugated streptavidin (Invitrogen) and the chromogenic reaction developed using liquid DAB substrate pack (Biogenex, San Ramon, CA) according to the manufacturer’s instructions. β-catenin and cyclin-D1 stained cells from random and non-overlapping fields were counted under a light microscope, magnification of 200×
[31 (link)].
[31 (link)].
3
Immunohistochemical Analysis of Apoptosis and Proliferation Markers
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Immunostaining was performed as previously reported [30 ]. Briefly, 5-μm thin formalin-fixed and paraffin-embedded tumor sections were incubated with the rabbit anti-mouse/human caspase-3, Bax, TUNEL, PCNA (proliferating cell nuclear antigen), Bcl-2, and CD31, respectively, overnight at 4 °C. The antibody concentration was 1:600. The samples were then labeled with horseradish peroxidase-conjugated streptavidin (Invitrogen) and the chromogenic reaction was developed using the Liquid DAB Substrate Pack according to the manufacturer’s instructions. The stained cells from random and nonoverlapping fields were counted under a magnification of ×100 or ×400. Meanwhile, the sections were stained with hematoxylin and eosin for microscopic examination. All sections were observed in a blinded fashion and were photographed using a 400× normal light microscope.
4
Immunohistochemical Analysis of EMT Markers
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4 μm-thin formalin fixed and paraffin-embedded slides were incubated with the rabbit anti-mouse ZEB1, Vimentin, E-cadherin, N-cadherin, TGF-βand SMAD-7 (Bioworld Technology, Dublin, OH, USA), respectively overnight at 4°C. Antibody concentration was 1:1000. The samples were then labeled with horseradish peroxidase-conjugated streptavidin (Invitrogen) and the chromogenic reaction that was developed using Liquid DAB Substrate Pack according to the manufacturer’s instructions. The stained cells from random and non-overlapping fields were counted under a magnification of × 400 [15 (link),26 ].
5
Immunohistochemical Analysis of E-cadherin and Vimentin
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Briefly, 5-μm-thick formalin-fixed and paraffin-embedded slides were incubated with a rabbit anti-mouse/human E-cadherin (20874-1-AP) (Proteintech, USA) and anti-mouse/human vimentin (5741s) (Cell Signaling Technology, USA) overnight at 4°C. The samples were then labeled with horseradish peroxidase-conjugated streptavidin (Invitrogen), and the chromogenic reaction was developed using a liquid diaminobenzidine (DAB) substrate pack according to the manufacturer’s instructions. The stained cells from random and non-overlapping fields were counted under a magnification of ×200.39 (link),40 (link)
6
Influenza Virus Receptor Affinity Assay
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Receptor affinity was determined in a solid-phase direct virus-binding assay as previously described. In brief, influenza viruses were bound to fetuin-coated plates at 4 ˚C overnight. Biotinylated glycans (a2,3’SL; a2,6’SL; or a2,6’SLN; Glycotech Corporation, Gaithersburg, MD, USA) were added to influenza-coated plates at varying dilutions and incubated for 4 h. Glycan binding was detected by adding horseradish peroxidase-conjugated streptavidin (Invitrogen, Carlsbad, CA, USA) followed by 3,3′,5,5′-tetramethylbenzidine substrate (Sigma-Aldrich, St Louis, MO, USA); the resulting absorbance at 450 nm was measured in a VICTOR3 1420 multilabel-counter plate reader (Perkin-Elmer, Waltham, MA, USA).
7
Cytokine Quantification by ELISA
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Levels of GM-CSF, IFN-γ, IL-4 and IL-10 on culture supernatants were assessed by ELISA. For IFN-γ, IL-4 and IL-10, ELISA kits from BioLegend San Diego, CA, USA were used according to the manufacturer’s instructions. GM-CSF concentration in the supernatants was measured using a purified anti-GM-CSF mAb (BVD2-23B6, BioLegend, San Diego, CA, USA) as capture antibody and a biotinylated anti-GM-CSF mAb (BVD2-21C11, BioLegend, San Diego, CA, USA) as detection antibody. Signal was detected by incubating plates with horseradish peroxidase-conjugated streptavidin (Invitrogen, Eugene, Oregon, USA), followed by o-phenylenediamine dihydrochloride (OPD) substrate (Sigma, St. Louis, MO, USA). Absorbances were read using a Bio-Tek (Winooski, Vermont, USA) uQuant microplate reader and the Gen5 software.
8
Protein Expression Detection in Cell Samples
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Cultured cells were lysed with a 1×sample buffer. Conditioned medium, cultured medium and the samples lysed with TBS buffer were mixed with a 2×sample buffer and boiled for 5 min. Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (10600001; Cytiva). The following antibodies were used: anti-ß-actin (C-15) mouse monoclonal antibody (mAb) (A5441; Merck), MK (A-9) mouse mAb (sc-46701; Santa Cruz Biotechnology), RPS6 (5G10) rabbit mAb (#2217; CST), phospho-RPS6 (D57.2.2E) XP rabbit mAb (#4858; CST), insulin-like growth factor binding protein 2 (IGFBP2) (EPR18012-257) rabbit mAb (ab188200; Abcam). ALK (C26G7) rabbit mAb (#3333; CST), ApoER2 (EPR3326) rabbit mAb (ab108208; Abcam), Syndecan-2 rabbit antibody (#36-6200; Invitrogen), Horseradish Peroxidase-conjugated streptavidin (434323; Invitrogen) and peroxidase-conjugated rabbit anti-mouse IgG or goat anti-rabbit IgG (315-035-048 or 111-035-144; Jackson ImmunoResearch). Chemiluminescence was developed using the Immobilon Classico or Forte substrate (WBLUC0100 or WBLUF0100; Merck) and detected on the Amersham Imager 680 (Cytiva). Silver staining was conducted using PierceTM Silver Stain Kit (24612; Thermo Fisher Scientific Inc. Inc.) according to manufactures protocol.
9
Sialylglycopolymer Binding Assay for Influenza Viruses
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Flat-bottom, 96-well immune assay plates (ThermoFisher) were coated with 10 µg/mL fetuin (Sigma-Aldrich) at 4 °C overnight then washed three times with washing buffer (PBS with 0.01 % Tween 80). Plates were blocked with PBS containing 1 % BSA then incubated at 4 °C overnight with 32 HA units of the Wigeon/SC/21 A/(H5N1), Eagle/FL/22 A/(H5N1), or CA/04 A/(H1N1)pdm09 viruses. Plates were incubated with biotinylated sialylglycopolymers: 3’-SialLacNAc-PAA-biotin (3’-SLN) (Glycotech) or 6’-SialLacNAc-PAA-biotin (6’-SLN) (Glycotech), serially diluted in the reaction buffer (PBS with 0.02% Tween 80, 0.02 % BSA, and 5 µM oseltamivir carboxylate). After incubation for 2 h at 4 °C, the plates were washed and incubated for 1 h with horseradish peroxidase-conjugated streptavidin (Invitrogen; diluted 1:2000) in blocking solution. After washing, the plates were incubated with o-phenylenediamine dihydrochloride (Sigma-Aldrich) at room temperature for 10 min. The reaction was stopped by adding 1 N H2SO4 (Fisher Chemicals), and the absorbance was measured at 490 nm in a Synergy H1 microplate reader (BioTek).
10
Immunohistochemical Analysis of Ovarian Tissue
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Paraffin-embedded ovarian tissue sectioned at 4 µm, mounted on slides and subjected to immunohistochemistry as described previously [65] (link). Sections were incubated at 4°C with polyclonal antibodies against PCNA (Santa Cruz sc-56), cytokeratin 8 (Developmental Studies Hybridoma Bank, TROMA I), ERα (Novacastra Laboratories), p-Akt1/2/3 serine 473 (Santa Cruz SC-33437), AMH (Santa Cruz Biotechnology SC-6886), WT1 (Santa Cruz Biotechnology), PAX8 (Proteintech group 10336-1-AP), calretinin (Invitrogen 18-0291), Ber-EP4 (Dako), aromatase (Abcam ab35604), vimentin (Sigma Aldrich V5255). Biotinylated secondary antibodies were used followed by incubation with horseradish peroxidase-conjugated streptavidin (Invitrogen). Sections were stained in AEC Solution.
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