To determine the subpopulation of 28-4+ NK cells in IEL cells from the duodenum tissue of control and NDV infected chickens, the isolated IEL cells were stained with 28-4 mAb. Briefly, 2 million cells/ml were re-suspended in 100 μl cold PBS mixed with 10 μl 28-4 Mab, incubated, and shaken on ice for 30 min. After centrifugation, the supernatant was removed and 100 μl cold PBS mixed with 12 μl Goat-Anti-Mouse-IgG3 (Abcam, USA) and then incubated with shaking on ice for 1 h. Furthermore, to check the purity of isolated CD3−/28-4+ IEL-NK cells, the cells were co-stained as described in the enrichment section. Cells were then washed and, 2 ml of 1% (v/v) buffered formalin (Sigma, Aldrich, USA) was mixed with the stained cells for immunophenotyping and kept at 4°C in dark foil. FACSCalibur™ flow cytometer (BD Biosciences, USA) and CellQuest Pro™ software (BD Biosciences, USA) were used in analyzing the results.
Goat anti mouse igg3
Manufactured by Abcam
Sourced in United Kingdom
Goat-Anti-Mouse-IgG3 is a secondary antibody that recognizes the IgG3 subclass of mouse immunoglobulins. It is designed for use in various immunological techniques, such as ELISA, Western blotting, and immunohistochemistry, where detection of mouse IgG3 is required.
Automatically generated - may contain errors
Lab products found in correlation
Buffered formalin, by Merck Group (1 mentions)
Cellquest pro software, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Goat anti mouse igg2a, by Abcam (1 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
Goat anti mouse igg1, by Abcam (1 mentions)
Spark reader, by Tecan (1 mentions)
Goat anti mouse igg horseradish peroxidase hrp, by Merck Group (1 mentions)
Goat anti mouse igg1 hrp, by Abcam (1 mentions)
Goat anti mouse igg2b hrp, by Abcam (1 mentions)
Goat anti mouse igg2a hrp, by Abcam (1 mentions)
Fitc anti mouse cd19, by BioLegend (1 mentions)
3 protocols using goat anti mouse igg3
1
Quantifying Duodenal IEL-NK Cells in Chickens
2
Chikungunya Virus IgG Isotype ELISA
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ELISA Nunc-Immuno 96-well MaxiSorp ELISA plates (Thermo Fisher Scientific, Darmstadt, Germany) were coated with 106 PFUs CHIKV per well in 100 μL PBS at 4°C overnight. After blocking for 1 h with 1% BSA in PBS, serum samples were serially diluted (1:50, 1:250, 1:1,000, and 1:5,000) for IgG endpoint titer determination, or for isotype determination, serum samples were diluted 1:100 in PBS containing 0.05% Tween 20 and 1% BSA, and incubated for 1 h at 37°C. The following anti-mouse secondary HRP-coupled antibodies were then added for 1 h at room temperature: rabbit anti-mouse total IgG (1:2,000; dianova, Hamburg, Germany; no. 115-035-003), goat-anti-mouse IgG1 (1:5,000; Abcam, Cambridge, UK; no. ab97240), goat-anti-mouse IgG2a (1:5,000; Abcam; no. ab97245), goat-anti-mouse IgG2b (1:5,000; Abcam; no. ab97250), or goat-anti-mouse IgG3 (1:5,000; Abcam; no. ab97260). For detection, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Merck Millipore, Darmstadt, Germany) was added, and the reaction was stopped after 15 min with 1 M H2SO4. Absorbance was measured at 450 nm (reference wavelength 620 nm) with a Tecan spark reader (Tecan, Männedorf, Switzerland).
3
Antibody Sources for Murine Immunoassays
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Antibodies used in ELISAs for IgG, IgG subclasses, and IgE were purchased as follows: goat anti-mouse IgG-horseradish peroxidase (HRP) was purchased from Sigma-Aldrich (St Louis, Mo), and the following antibodies were all purchased from Abcam (Cambridge, United Kingdom): goat anti-mouse IgE-HRP, goat anti-mouse IgG 1 -HRP, goat anti-mouse IgG 2a -HRP, goat anti-mouse IgG 2b -HRP, and goat anti-mouse IgG 3 . Antibodies for flow cytometric analyses were purchased from BioLegend (San Diego, Calif), as follows: anti-mouse CD45 in fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin, and peridinin-chlorophyllprotein complex/Cy5.5 and anti-mouse CD19-FITC, anti-mouse CD124-PE, and mouse isotype controls in FITC, PE, allophycocyanin, and peridinin-chlorophyll-protein complex/Cy5.5. Antibody against serotype 19 pneumococcal strains (Rb anti-S pneumoniae serotype 19) was provided by Dr Moon Nahm and Dr Brady Spencer (University of Alabama at Birmingham, Birmingham, Ala) for this work. Total IgE levels were examined by using a specific kit for Mouse IgE (BioLegend), according to the manufacturer's instructions. M pneumoniae P1-adhesin carboxy terminus (recombinant P1 [rP1]) peptide was purchased from ProSpec Biosciences (Ness-Ziona, Israel). Grade V ovalbumin (OVA) was purchased from Sigma-Aldrich for immunization, airway challenge, and enzyme immunoassay.
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