To assay sPD-L1 variants, 0.1 µg/well of mouse anti-human PD-L1 mab (130021, R&D systems) or 0.2 µg/well anti-human PD-L1 mAb (29E.12B1) were coated on Costar ELISA plates overnight at 4°C. Plates were then washed with PBS and blocked with protein-free blocking buffer (Pierce, Rockford, IL) for 4 hours. Patient sera or plasma were diluted with PBS in 1:1 volume ratio. 100 µl per well of diluted patient sera or plasma were added and incubated overnight at 4°C. Plates were washed with PBS containing Tween-20, and incubated with 100 µl per well of 0.1µg/well biotinylated anti-PD-L1 mAb (29E.2A3, Biolegend) in protein-free blocking buffer at room temperature for 2 hours. Plates were then washed and incubated with 1mg/ml streptavidin-HRP (Jackson ImmunoResearch) diluted 1:40,000 in protein-free blocking buffer for 2 hours. Plates were washed and treated with biotinyl tyramide (Perkin Elmer) for 30 min, and then washed and incubated with 1 mg/ml streptavidin-HRP (Jackson ImmunoResearch) diluted 1:400,000 in protein-free blocking buffer for 2 hours with further development with TMB (Pierce). Plates were read at an optical density (O.D.) of 450 nm. All samples were assayed in duplicate. A standard curve using recombinant human PD-L1-HIS (Novoprotein, Summit, NJ) was also performed with each assay.
Streptavidin hrp
Manufactured by Jackson ImmunoResearch
Sourced in United States, Panama
Streptavidin-HRP is a conjugate of the protein streptavidin and the enzyme horseradish peroxidase (HRP). Streptavidin has a high affinity for the small molecule biotin, while HRP catalyzes colorimetric or chemiluminescent reactions. This conjugate can be used to detect and quantify biotinylated proteins, nucleic acids, or other molecules in various assays.
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Lab products found in correlation
Microlon f shape ref 655092, by Greiner (3 mentions)
Maxisorp plate, by Thermo Fisher Scientific (3 mentions)
Goat anti human igg hrp, by Jackson ImmunoResearch (3 mentions)
Anti neu5gc igy, by BioLegend (3 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Supersignal pico chemiluminescent reagent, by Thermo Fisher Scientific (2 mentions)
Aal biotin, by Vector Laboratories (2 mentions)
Ultra tmb elisa substrate solution, by Thermo Fisher Scientific (2 mentions)
Biotinylated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Prism 10, by GraphPad (2 mentions)
Aec substrate, by BD (2 mentions)
Casein, by Thermo Fisher Scientific (2 mentions)
Spectramax 340pc, by Molecular Devices (2 mentions)
Aec substrate, by Merck Group (2 mentions)
Anti human ig, by Jackson ImmunoResearch (2 mentions)
Anti human igm biotin, by Thermo Fisher Scientific (2 mentions)
Igg igadouble color elispot kits, by Cellular Technology (2 mentions)
Anelispot counter, by Cellular Technology (2 mentions)
Elispot plate, by Merck Group (2 mentions)
Hematoxylin, by Merck Group (2 mentions)
81 protocols using streptavidin hrp
1
Quantifying soluble PD-L1 variants
2
Immunohistochemical Analysis of Joint Proteins
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Immunohistochemistry was performed using anti-αSMA (Abcam, ab5694, 1:200), anti-MMP2 (R and D Systems, AF1488, 1:400), and anti-phospho-NFκB p65 (Ser536) (Cell Signaling (93H1), #3033, 1:20) antibodies. Briefly, slides of knee joints from 4 month wildtype and Crtap-/- mice were deparaffinized and treated with 3% hydrogen peroxide and proteinase K solution (20 mg/ml) for 10 min. Slides were blocked using 5% normal donkey serum and incubated for 1 hr. Primary antibodies were diluted in blocking solution and incubated overnight at 4°C. Slides were washed with PBS and incubated in anti-rabbit-biotin (Jackson Immunoresearch, 711-065-152, 1:400 for αSMA or 1:100 for phospho-NFκB) or anti-goat-biotin (Jackson Immunoresearch, 705-065-147, 1:500 for MMP2) for 1 hr. Following PBS wash, slides were incubated in streptavidin-HRP (Jackson Immunoresearch, 016-030-084, 1:400–500 for αSMA and MMP2 or 1:100 for phosho-NFκB) for 30 min then washed with PBS. Slides were incubated with DAB substrate (Vector Laboratories, SK-4100) for approximately 2 min, counterstained with hematoxylin, then cleared and mounted with a coverslip.
3
Immunohistochemistry of FFPE Brain Tumor Samples
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FFPE tissue from surgically resected MB patient samples was obtained with informed consent and following the ethical regulations at the University of Manitoba, the Hospital for Sick Children and the University of Calgary’s Clark Smith Brain Tumour Bank. MB subgroup classification was previously determined by NanoString profiling analyses68 (link). Following deparaffinization of FFPE patient or xenograft samples, antigen retrieval was performed in citrate buffer at 95–100 °C for 20 min. Slides were washed and treated for 10 min for endogenous peroxidase, and again washed in 1× PBS. The samples were blocked with 3% lamb serum, then incubated with primary antibodies (Supplementary Table 1 ) overnight at 4 °C. Slides were treated with secondary antibody (Supplementary Table 1 ) for 2 h at room temperature. Streptavidin/HRP (Jackson ImmunoResearch, West Grove, PA, USA) in 1× PBS was then added for 30 min followed by development with DAB and counterstaining with hematoxylin. Finally, coverslips were mounted with Permount (Fisher Scientific).
4
Histological Examination of Feline Intestinal Tissue
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For histological studies, infected cats were euthanized at day 6/7 and portions of intestinal ileum were fixed in 10% buffered neutral formalin. Fixed tissues were cut into sections (2.5 x 0.7 cm), placed in cassettes, embedded in paraffin, and sectioned 4–5 μm thick. Slides were deparaffinized, rehydrated, and stained with hematoxylin and eosin (Leica Microsystems, Buffalo Grove, IL), or by immunohistochemistry with Rabbit anti-RH polyclonal antibody [39 (link)] and Streptavidin-HRP (Jackson Labs, West Grove, PA), according to standard protocols [1 ].
5
Quantifying AID Protein Levels
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To measure AID protein levels, 5 × 106 cells were lysed in 0.5 mL RIPA buffer (1% NP-40, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) for 1 h at 4 °C. Fifty μg of total protein from the clarified lysates were separated on a 12.5% reducing SDS-PAGE, transferred to nitrocellulose paper and sequentially stained with biotinylated goat anti-HA antibody (Vector Laboratories, CA, USA) or rabbit anti-tubulin α antibody (NeoMarkers, CA, USA) followed by streptavidin-HRP and goat anti-rabbit Ig-HRP, respectively (Jackson ImmunoResearch Laboratories, PA, USA). Bands were visualized by ECL detection (Thermo Fisher Scientific) and analyzed with a LAS-3000 Mini Fujifilm imaging system (FujiFilm, Tokyo, Japan).
6
Western Blotting Protocol for Protein Detection
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Western blotting was performed according to standard methods using HRP-coupled or IRDye (LI-COR Biosciences) secondary antibodies (all antibodies are listed in Table S4 ). Biotinylated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and blocked with 3% BSA in TBS-T (24.8 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 1% (v/v) Tween 20) overnight at 4 °C. Membranes were incubated with streptavidin-HRP (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; diluted 1:5000–1:40,000) for 1 h at room temperature and washed three times with TBS-T. To detect proteins, Immobilon Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA) or the LI-COR Odyssey Imaging System was used.
7
Western Blot Analysis of Pontin Protein
8
ELISA Protocol for Cytokine Quantification
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Cytokine concentrations in culture supernatants were determined by ELISA, using cytokine-specific coating and biotinylated detection antibodies diluted per manufacturer’s protocol (eBioscience/Invitrogen/Thermo Fisher Scientific). Streptavidin-HRP (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) binding to biotinylated detection antibodies was visualized with TMB substrate (KPL/Seracare, Milford, MA) and the reaction was stopped with 0.18 M H2SO4. Plates were read at 450 nm via Emax Plus Reader (Molecular Devices, San Jose, CA). Data were analyzed with SoftMax Pro software (Molecular Devices); unknowns were compared with a standard curve containing at least five to seven dilution points of the relevant recombinant cytokine (eBioscience/Invitrogen/Thermo Fisher Scientific) on each assay plate. In all cases, the coefficient of determination for the standard curve (r2) was ≥0.98. ELISA unknowns were diluted to fall within the range of standard values.
9
Quantifying IFN-γ Release from Activated YTS Cells
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To assess the activation capacity of the YTS cells with the stimulus of CD48 positive (721.221, BCBL1) and negative (K562) target cells, YTS cells were incubated for 48h at 1:1 and 1:0.5 ratios with 50,000 effector cells at 37°C. The cell-free supernatant was used for IFN-γ specific sandwich ELISA. Nunc MaxiSorp™ flat-bottom ELISA plates (Invitrogen) were coated with 1μg/ml purified anti-IFN-γ (BLG-502402, Biolegend) in 50μl PBSx1 and incubated for 2h at 37°C followed by blocking with 200μl 1% BSA in PBSx1 incubated for 2h at room temperature (RT). Washing buffer of PBSx1 + 0.05% Tween-20, was used for washing the wells 3 times. 100μl of supernatant were incubated within the coated wells at 4°C overnight. The biotinylated IFN-γ detection antibody (BLG-502504, Biolegend) was then added at 1μg/ml in 100μl 1% BSA in PBSx1 and incubated for 1h at RT. Finally, streptavidin HRP (016-030-084, Jackson immuno research) 1μg/ml in 100 μl PBSx1 + 0.05% Tween-20 + 1% BSA was incubated for 30 min at RT, and quantification was performed with TMB one component substrate (Southern Biotech).
10
TNF-alpha Quantification by ELISA
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