A sandwich ELISA was used to determine the levels of IL-6 and TNF-α cytokines in sera of IFN-I signal-competent and -incompetent mice following JEV infection. ELISA plates were coated with IL-6 (MP5-20F3), TNF-α (1F3F3D4) anti-mouse Ab purchased from eBioscience, and then incubated overnight at 4 °C. The plates were washed three times with PBS containing 0.05% Tween (PBST), after which they were blocked with 3% bovine serum albumin for 2 h at 37 °C. The sera and standards for recombinant cytokine proteins (PeproTech, Rocky Hill, NJ) were added to the plates, which were then incubated for 2 h at 37 °C. The plates were washed again with PBST, and biotinylated IL-6 (MP5-32C11) and TNF-α (MP6-XT22) Ab were added. Next, the plates were incubated overnight at 4 °C, followed by washing with PBST and subsequent incubation with peroxidase-conjugated streptavidin (eBioscience) for 1 h. Color development was then performed by the addition of a substrate (ABTS) solution. Cytokine concentrations were determined with an automated ELISA reader and SoftMax Pro3.4 according to comparisons with two concentrations of standard cytokine proteins.
Streptavidin conjugated peroxidase
Manufactured by Thermo Fisher Scientific
Sourced in United States
Streptavidin-conjugated peroxidase is a laboratory reagent used in various immunoassay techniques. It consists of the protein streptavidin, which has a high affinity for the biomolecule biotin, covalently linked to the enzyme horseradish peroxidase. This conjugate can be used to detect and quantify biotinylated targets in samples, such as proteins or nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Standards for recombinant cytokine proteins, by Thermo Fisher Scientific (2 mentions)
3 3 diaminobenzidine (dab), by Thermo Fisher Scientific (2 mentions)
Recombinant cytokine proteins, by Thermo Fisher Scientific (1 mentions)
Histostain plus ihc kit, by Thermo Fisher Scientific (1 mentions)
5 bromo 2 deoxyuridine brdu, by Merck Group (1 mentions)
Mouse monoclonal anti brdu, by BD (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
Dab substrate, by Vector Laboratories (1 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
Tmb microwell peroxidase substrate system, by LGC (1 mentions)
Blocking one solution, by Nacalai Tesque (1 mentions)
Biotin conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti 8 ohdg antibody, by Santa Cruz Biotechnology (1 mentions)
Biotin conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
10 protocols using streptavidin conjugated peroxidase
1
Sandwich ELISA for Cytokine Detection
2
Cytokine Quantification by Sandwich ELISA
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Sandwich ELISA was used to determine cytokine levels in culture supernatants. ELISA plates were coated with TNF-α (1F3F3D4), IL-4 (11B11), IL-5 (TRFK5), IL-6 (MP5-20F3), IL-10 (JES5-16E3), and IFN-γ (R4-6A2) antibodies (eBioscience or BD Bioscience) and incubated at 4°C overnight. After plates were washed three times with PBS containing 0.05% Tween 20 (PBST), they were blocked with 3% nonfat dried milk at 37°C for 2 h. Culture supernatant and standards for recombinant cytokine proteins (Peprotech, Rehovot, Israel) were added to these plates and incubated at 37°C for 2 h. Plates were washed again with PBST and biotinylated TNF-α (polyclonal antibody), IL-4 (BVD6-24G2), IL-5 (TRFK4), IL-6 (MP5-32C11), IL-10 (JES5-2A5), and IFN-γ (XMG1.2) antibodies were added. The mixture was incubated overnight at 4°C followed by washing with PBST and subsequent incubation with peroxidase-conjugated streptavidin (eBioscience) at 37°C for 1 h. Color was then developed by adding a substrate (ABTS) solution. Cytokine concentrations were determined using an automated ELISA reader and SoftMax Pro4.3 by comparison with two concentrations of standard cytokine proteins.
3
Quantifying IL-6 and TNF-α Cytokines by Sandwich ELISA
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Sandwich ELISA was used to determine the levels of IL-6 and TNF-α cytokines in sera. ELISA plates were coated with IL-6 (MP5-20F3) and TNF-α (1F3F3D4) antibodies (eBioscience), and incubated at 4°C overnight. After plates were washed three times with PBS containing 0.05% Tween 20 (PBST), they were blocked with 3% non-fat-dried milk at 37°C for 2 h. Sera and standards for recombinant cytokine proteins (Peprotech, Rehovot, Israel) were added to these plates and incubated at 37°C for 2 h. Plates were washed again with PBST, and then biotinylated IL-6 (MP5-32C11) and TNF-α (polyclonal antibody) antibodies were added. The mixture was incubated overnight at 4°C followed by washing with PBST and subsequent incubation with peroxidase-conjugated streptavidin (eBioscience) at 37°C for 1 h. Color was then developed by adding a substrate (ABTS) solution. Cytokine concentrations were determined using an automated ELISA reader and SoftMax Pro4.3 by comparison with two concentrations of standard cytokine proteins.
4
Craniofacial Embryonic Development Protocol
5
Quantitative Immunohistochemistry for PHF14 Expression
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Tissues were processed into paraformaldehyde-fixed, paraffin-embedded specimens. Sections were incubated with anti-PHF14 (produced in our own laboratory) followed by biotinylated secondary antibodies and peroxidase-conjugated streptavidin (Thermo Fisher Scientific). Finally, the sections were counterstained with hematoxylin and examined. Positivity for PHF14 positive cells and staining intensity was assessed semi-quantitatively by three independent investigators without prior knowledge of the clinical follow-up data. Cases were accepted as positive only if reviewers independently defined them as such. The percentage of positive cells was rated as follows: 1, 1-10% positive cells; 2, 11-50% positive cells; 3, 51-75% positive cells; and 4, >75% positive cells. Staining intensity was scored as follows: 1, negative; 2, weak; 3, moderate; and 4, intensive. The scores for the percentage of positive cells and the scores for expression intensity were multiplied to calculate an immunoreactive score (IRS): 1-4 +/−; 5-8 = weak staining; 9-12 = moderate staining; and 13-16 = strong staining.
6
Quantification of Soluble RCAS1 by ELISA
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Soluble-form RCAS1 in SiSo Sup was measured by ELISA. Briefly, for precoating, 22-1-1 antibody (Medical & Biological Laboratories Co., Ltd.) was added to each well in a 96-well plate (100 µl/well) and incubated overnight at 4°C. After washing with PBS, Blocking One solution (Nacalai Tesque, Inc.) was diluted 5-fold with PBS and added to each well for blocking. After 1 h of incubation at room temperature, diluted SiSo Sup was added and incubated for 1 h at room temperature. After washing, biotinylated 22-1-1 antibody was added and incubated for 1 h at room temperature. After washing, peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Inc.) was added and incubated for 1 h at room temperature. Then, peroxidase substrate solution (TMB Microwell Peroxidase Substrate System Kirkegaard & Perry Laboratories, Inc.) was added to each well and incubated for 20 min at room temperature, followed by an equal volume of 1 M H3PO4. Absorbance was measured at 450 nm (reference wavelength: 655 nm) with a microplate reader (iMark Microplate Reader; Bio-Rad Laboratories, Inc.).
7
Immunohistochemical Analysis of Oxidative Stress
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The immunohistochemistry staining methods were performed as previously described35 (link). After dewaxing, antigen retrieval and blocking of endogenous peroxidase, the sections were fixed with goat serum and incubated with a 1:1000 dilution of rabbit monoclonal anti-CGL antibody (Abcam, Cambridge MA, USA), a 1:2000 dilution of rabbit monoclonal anti-CBS antibody (Abcam, Cambridge MA, USA), and a 1:20 dilution of mouse monoclonal anti-8-OHdG antibody (Santa cruz biotechnology, CA, USA) overnight at 4 °C, followed by incubation with biotin-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) and streptavidin- conjugated peroxidase (Invitrogen, Carlsbad, CA, USA). Finally, sections were detected under a microscope after reaction using 3,3′-diaminobenzidine (DAB) (Invitrogen, Carlsbad, CA, USA). The intensity of immunohistochemical staining of 8-OHdG was analyzed in ten random fields (×400) of renal cortex per rat using Image J software (NIH, MD, USA).
8
Immunohistochemical Analysis of TLR2 in Kidney Tissue
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The kidneys were fixed in 10% formaldehyde overnight at 4°C and processed for paraffin-embedding according to standard procedures. Sections were cut at 3-µm thicknesses. For immunohistochemical analyses, some tissue sections were subjected to antigen retrieval by microwaving or autoclaving for 10 or 15 min in 10 mM sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by a 10-min incubation with 3% hydrogen peroxide. Sections were washed with PBS and subsequently incubated with 1.5% normal goat serum for 20 min, followed by overnight incubation with primary 1∶200 anti-TLR2 antibody at 4°C. After 3 washes with PBS, the samples were incubated with biotin-conjugated anti-IgG for 30 min at room temperature. After another wash in PBS, the sections were then incubated with a streptavidin-conjugated peroxidase (Invitrogen Corp., Carlsbad, CA, USA) for 30 min at room temperature. After a final wash in PBS, the sections were incubated with diaminobenzidine (Invitrogen) followed by microscopic examination.
9
Immunohistochemical Analysis of Kidney Tissue
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Kidneys were fixed in 10% formaldehyde overnight at 4°C and processed for paraffin embedding according to standard procedures. Sections were cut at 3-μm thickness. For immunohistochemical analyses, some tissue sections were subjected to antigen retrieval by microwaving or autoclaving for 10 or 15 min in 10 mM sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by a 10-min incubation in 3% hydrogen peroxide. Sections were washed with phosphate-buffered saline (PBS) and subsequently incubated with 1.5% normal goat serum for 20 min, followed by overnight incubation with primary antibodies (1:50 anti-CD68 antibody; 1:200 anti-TLR2 and anti-TLR4 antibodies) at 4°C. After three washes with PBS, the samples were incubated with biotin-conjugated anti-IgG for 30 min at room temperature. After another wash in PBS, the sections were incubated with a streptavidin-conjugated peroxidase (Invitrogen) for 30 min at room temperature. After a final wash in PBS, the sections were incubated with diaminobenzidine (Invitrogen) followed by microscopic examination.
10
Kidney Tissue Immunohistochemistry Protocol
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The kidneys were fixed in 10% formaldehyde overnight at 4°C and processed for paraffin-embedding following standard procedures. Sections were prepared at 2 mm thicknesses. For immunohistochemical analysis, some tissue sections were subjected to antigen retrieval by microwaving or autoclaving for 11 min in 10 mM sodium citrate buffer [pH 6.0]. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 15 min. After PBS washing, sections were incubated with 1.5% normal goat serum for 30 min, followed by incubation with mouse monoclonal anti-E-cadherin (1:50; Cambridge, MA) and rabbit polyclonal anti-α-SMA (1:100, Cambridge, MA) overnight at 4°C. After three washes with PBS, the samples were incubated with biotin-conjugated goat anti-mouse IgG (Invitrogen Corporation, CA, USA) for 40 min at room temperature. After washing in PBS, the sections were incubated with streptavidin-conjugated peroxidase (Invitrogen Corporation, CA, USA) 30 min at room temperature. After PBS washing, the sections were incubated with DAB (Invitrogen Corporation, CA, USA) followed by examination under the microscope.
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