Mice were immunized subcutaneously with OVA (Sigma) in CFA (Sigma) and euthanized after 14 days. Blood was collected and allowed to coagulate in the refrigerator in Microtainer Serum Separator tube (BD). Serum was isolated by centrifugation. For ELISA to detect OVA-specific Ig, plates were coated with 2 mg ml−1 OVA. Antibody levels in serially diluted serum samples were analysed with HRP-conjugated antibodies (1:4,000) specific for mouse IgM (1021-05), IgG (1030-05), IgG1 (1070-05), and IgG2c (1079-05) (SouthernBiotech) following manufacture's recommended protocols. For ex vivo cytokine production, splenocytes were isolated 14 days following OVA-CFA immunization and cultured in the presence of OVA (100 μg ml−1). ELISAs were performed with purified anti-IFNγ (Biolegend 505707) and anti-IL-4 mAb (Biolegend 504102) as capture antibodies, the corresponding biotinylated anti-IFNγ (Biolegend 505804, 1 μg ml−1)and anti-IL-4 mAb (BD Biosciences 554390 1 μg ml−1), horseradish peroxidase-conjugated streptavidin (Sigma), and the TMB microwell peroxidase substrate and stop solution (Kirkegaard & Perry Labs, Inc.) according to the recommended protocol (Biolegend). Recombinant mouse IFNγ and IL-4 (R&D Systems) was used as standards.
Horseradish peroxidase conjugated streptavidin
Manufactured by Merck Group
Sourced in United States
Horseradish peroxidase-conjugated streptavidin is a protein complex composed of streptavidin, a tetrameric protein that binds to biotin, and horseradish peroxidase, an enzyme that catalyzes a colorimetric reaction. This complex can be used as a detection or labeling reagent in various bioanalytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Complete freund adjuvant (cfa), by Merck Group (2 mentions)
Sunrise absorbance reader, by Tecan (2 mentions)
Biotinylated secondary antibody, by Vector Laboratories (2 mentions)
Versadoc 5000 imager, by Bio-Rad (2 mentions)
Chemi lumi one, by Nacalai Tesque (2 mentions)
Microtainer serum separator tube, by BD (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Hrp conjugated antibodies, by Southern Biotech (1 mentions)
Recombinant mouse ifn γ and il 4, by R&D Systems (1 mentions)
Dab substrate, by Thermo Fisher Scientific (1 mentions)
Image pro plus, by Thermo Fisher Scientific (1 mentions)
Prism 9, by GraphPad (1 mentions)
P8340, by Merck Group (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Nupage bis tris precast polyacrylamide gels, by Thermo Fisher Scientific (1 mentions)
Anti gapdh horseradish peroxidase, by Merck Group (1 mentions)
Dab reaction, by Merck Group (1 mentions)
Nanozoomer slide scanner, by Hamamatsu Photonics (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
O phenylenediamine dihydrochloride peroxidase substrate, by Merck Group (1 mentions)
18 protocols using horseradish peroxidase conjugated streptavidin
1
OVA-Specific Antibody and Cytokine Responses
2
Immunohistochemical Quantification of Angiogenic Markers
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Tumor samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned into 4-μm-thickness slices. After dewaxing, tissues sections were processed by antigen retrieval, followed by the quenching of endogenous peroxidase activity using hydrogen peroxide. PBS (0.1% Tween 20) containing normal serum was used to block non-specific binding sites. Slides were incubated with rabbit polyclonal antibodies against CD31 or VEGF-A (in 1:50 dilution, Santa Cruz, US). After washing with PBS, slides were incubated with biotinylated anti-rabbit IgG antibody followed by horseradish peroxidase-conjugated streptavidin (Sigma, US). After developing in DAB substrates (Invitrogen, US), 5 high-magnification fields (400×) were randomly selected in each slide, and the numbers of positive cells in each field were counted by the image analysis system (Image-Pro Plus). In a parallel negative control group, PBS was applied to replace the primary antibody.
3
Characterization of CD86+ MARCH/MIR+ Cells
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HeLa cell lines generated for experiments in Figures 1 and 6 were flow sorted to generate populations of pure CD86+ MARCH/MIR+ cells, and replicate cell samples were cultured in 6-well tissue culture plates with or without 500 ng/ml DOX (Sigma) for 2 days. One well for each condition was lysed in 0.5 ml radioimmunoprecipitation assay buffer containing complete protease inhibitor (P8340; Sigma) for 1 h on ice and then cleared by centrifugation. Samples were mixed with 4× NuPAGE nonreducing lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific), bath sonicated, and heated at 95 °C for 5 min. Lysate samples were separated by electrophoresis in 12% NuPAGE Bis–Tris precast polyacrylamide gels (Thermo Fisher Scientific) at 200 V for 40 min in MES running buffer and transferred to polyvinylidene difluoride membranes that were then blocked in Tris-buffered saline with 0.1% Tween-20 containing 5% w/v bovine serum albumin. CD86 was detected using biotinylated anti-CD86 mAb IT2.2 (Biolegend) and horseradish peroxidase–conjugated streptavidin (Sigma). GAPDH was detected as a loading control using anti-GAPDH-horseradish peroxidase (Sigma). Quantification was performed in Image Lab (Bio-Rad Laboratories), and results were analyzed in Prism 9 (GraphPad Software).
4
Astrocyte and Microglia Response to AMPA
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2–4 month-old wild-type mice (n = 6 per group) or APP/PS1 mice (n = 3 per group) were treated with 8 h or 14 h, respectively, of AMPA or artificial CSF via reverse microdialysis then immediately transcardially perfused with ice-cold phosphate buffer saline (PBS) with 0.3% heparin. Brains were removed, fixed in 4% paraformaldehyde for 24 h at 4 °C, then placed in 30% sucrose prior to freezing and sectioning. Coronal brain sections 50 μm wide were sliced in 300 μm intervals using a freezing sliding microtome. Sections were then immunostained to visualize astrocytes or microglia using antibodies against glial fibrillary acidic protein (GFAP; 1:500, ThermoFisher) as an astrocytic marker or against ionized calcium-binding adaptor molecule 1 (Iba1; 1:500; Wako Laboratory Chemicals, Richmond, VA) as a microglial marker. Biotinylated secondary antibody, horseradish peroxidase-conjugated streptavidin, and DAB reaction (Sigma) were used to develop. Brain sections were imaged with a Nanozoomer slide scanner (Hamamatsu Photonics, Bridgewater, NJ). Staining density was qualitatively evaluated by blinded observers and vehicle- and AMPA-treated groups were compared. Images shown are representative.
5
Amyloid-β and HMGB1 Binding Assay
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Ig-fusion protein of sRAGE in a concentration of 5 mg/mL was bound to protein A (1 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) coated wells (Maxisorb ELISA plates, Sigma-Aldrich). Unbound proteins were washed away, and the wells were blocked with BSA (Sigma-Aldrich). Biotinylated amyloid β 1–42 peptide (20 nM, American Peptide, Sunnyvale, CA, USA) or biotinylated HMGB1 (20 nM) were bound to the wells in the presence or absence of inhibitors, and unbound ligands were washed away. Bound biotinylated ligands were detected with horse radish peroxidase conjugated streptavidin (Sigma-Aldrich) and peroxidase substrate (Sigma-Aldrich). The color was developed using o-phenylenediamine dihydrochloride peroxidase substrate (Sigma-Aldrich). Absorbance at 490 nm was measured.
6
Quantifying Surface Expression of TRPM6 and TRPM7
7
GST-UbcH10 Binding Assay
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Wells were coated overnight at 4°C with 100 µg/ml GST-UbcH10 in PBS 1X, 1 mM TCEP, in the presence of an EDTA free protease inhibitor cocktail (Roche Diagnostics). Binding step was performed with different concentrations of biotinylated peptides L1, L2, ScrL2, U1, ScrU1, S2 (2.2, 11, 22, 44, 108 µM) in PBS 1X (with 1 mM TCEP for U1 and ScrU1). A blocking solution 1% BSA in PBS 1X, 0.05% Tween-20 was used. Washes were executed with PBS 1X, 0.05% Tween-20. To verify the interaction a 1∶10000 dilution of horseradish peroxidase-conjugated streptavidin (Sigma Aldrich) in 0.3% BSA, PBS 1X was incubated for 1 hour. The colorimetric reaction was carried out with SIGMA-FAST OPD reagent (Sigma Aldrich), according to the manufacturer's instructions. Finally, readings were performed at 490 nm on a Model680 MicroplateReader (Bio-Rad, Hercules, CA-USA), and data were recorded by Microplate Manager 5.2 program and elaborated by GraphPad Prism program. Negative control experiments with the fusion tag GST in coating were performed in the same conditions described above.
8
Detecting Keap1 Oxidative Modifications
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The oxidized form of Keap1 harboring oxidative thiol modification was examined as previously reported [41 (link)]. In brief, kidney specimens were homogenized and cultured cells lysed in radioimmunoprecipitation assay buffer containing protease inhibitors and 20 mM N-ethylmaleimide to stabilize the sulfhydryl moieties in all proteins. Then the disulfide bonds were reduced by treating with 20 mM 2-dithiothreitol (DTT) and afterwards selectively labeled with 50 μM 3-N-maleimido-propionyl biocytin (MPB) after removing the unreacted DTT. To detect Keap1-specific disulfide moieties, the MPB-labeled proteins were immunoprecipitated by Keap1 antibody and immunoprecipitates subjected to immunoblot analysis by detection with horseradish peroxidase-conjugated streptavidin (Sigma-Aldrich).
9
Serum Antibody Measurement by ELISA
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Serum-specific IgE, IgG1 and IgG2a were determined by ELISA with the required antibodies purchased from BD Pharmingen (San Jose, CA, USA). Microtiter plates were coated with specific antigens for 2 hours at 37°C. After washing with PBST, plates were blocked with 2% BSA for 2 hours at room temperature. Sera were diluted (1:10 for IgE or 1:100 for IgGs) in PBST and incubated at room temperature for 2 hours. For IgE measurement, the plates were incubated with biotin-conjugated rat anti-mouse IgE (1:1000) for 2 hours at room temperature. Subsequently, horseradish peroxidase-conjugated streptavidin (1:4,000) (Sigma, St Louis, MI, USA) was added for 1 hour and developed by adding 2,2’-azino-bis(3-ethylbenzthiazoline-sulfonic acid (ABTS, Sigma) as substrate for 30 minutes. For IgG measurement, the plates were incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG1 or IgG2a (1:1000) for 2 hours at room temperature and developed by adding ABTS as above. Then, the optical density was analyzed on a Sunrise Absorbance Reader (TECAN, Austria) at 415 nm.
10
Serum Antibody Isotype Analysis
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Serum-specific IgE, IgG1 and IgG2a antibodies were determined by ELISA with the required antibodies purchased from BD Pharmingen (San Jose, CA, USA). Microtiter plates were coated with specific antigens for 2 hours at 37°C. After washing with PBST, plates were blocked with 2% BSA for 2 hours at room temperature. Murine sera were diluted (1:10 for IgE or 1:100 for IgGs) in PBST and incubated at room temperature for 2 hours. For IgE measurement, the plates were incubated with biotin-conjugated rat anti-mouse IgE (1:1000) for 2 hours at room temperature. Subsequently, horseradish peroxidase-conjugated streptavidin (1:4000) (Sigma) was added to the plates for 1 hour followed by addition of tetramethylbenzidine (TMB) as a substrate for 10 minutes. Finally, the reaction was stopped by adding 2M sulfuric acid. For IgG measurement, the plates were incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG1 or IgG2a (1:10000) for 2 hours at room temperature and developed by adding 2,2’-azino-bis(3-ethylbenzthiazoline-sulfonic acid (ABTS, Sigma). Then, the optical density was analyzed on a Sunrise Absorbance Reader (TECAN, Austria) at 450 or 415 nm, respectively.
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