Six weeks after the last immunization, sera were collected from vaccinated mice. WH121-specific end point titers for IgG1, IgG2c, and total IgG antibodies were detected by ELISA. Using previously described methods (48 (link)), microtiter plates were pre-coated with 100 µL WH121 protein (5 µg/mL) in carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C and blocked with 1% BSA in PBST for 1 h at 37°C. Horseradish peroxidase-conjugated rabbit anti-mouse IgG (1/5,000; Abcam, UK, Cat. no. Ab6789), IgG1 (1/10,000; Abcam, UK, Cat. no. Ab97240), or IgG2c (1/10,000; Abcam, UK, Cat. no. Ab97255) were added to the plates. 100 µL 3,3′5,5′-tetramethylbenzidine substrate was used for visualization at 37°C. Plates were read at 450 nm using a Multiskan ELISA reader (Bio-tek, USA). Antibody titers in each group were determined by comparison to the PBS control group. The results are expressed as mean (±SEM) log10 end point titers, and as the ratio of IgG2c/IgG1.
Horseradish peroxidase conjugated rabbit anti mouse igg
Manufactured by Abcam
Sourced in United Kingdom, Hong Kong
Horseradish peroxidase-conjugated rabbit anti-mouse IgG is a secondary antibody that binds to mouse IgG antibodies. It is designed for use in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti caspase 3 antibody, by Abcam (1 mentions)
Rabbit anti prx1 antibody, by Abcam (1 mentions)
Horseradish peroxidase conjugated swine anti rabbit igg, by Agilent Technologies (1 mentions)
Mouse anti gapdh, by Abcam (1 mentions)
Exendin 4, by Abcam (1 mentions)
Thermo multiskan ex plate reader, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
6 protocols using horseradish peroxidase conjugated rabbit anti mouse igg
1
ELISA for Antibody Titers in Vaccinated Mice
2
Western Blot Analysis of PRX1 and Caspase-3
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MC3T3-E1 cells were harvested at selected time points and lysed in RIPA lysis buffer (ComWin Biotech Co., Ltd., Beijing, China). Protein samples (50 μg) were mixed with 1/4 volume of 5 × SDS loading buffer and boiled at 95 °C for 5 min. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Immunoblotting was performed with the following antibodies: rabbit anti-PRX1 antibody (Abcam Ltd., Shanghai, China) at a dilution of 1:1000, rabbit anti-caspase-3 antibody (Abcam Ltd., Shanghai, China) at a dilution of 1:2000 and mouse anti-GAPDH (Abcam Ltd., Shanghai, China) at a dilution of 1:2000, overnight at 4 °C. The secondary antibodies used were horseradish peroxidase-conjugated swine anti-rabbit IgG (DaKo, Glostrup, Denmark) for PRX1 and caspase-3, and horseradish peroxidase-conjugated rabbit anti-mouse IgG (Abcam Ltd., Hong Kong) for GAPDH, at room temperature for 1h. Western blot images were captured using a FluorChem E System (ProteinSimple, Santa Clara, CA, USA). The blots were cropped from different gels but all the gels had been run under the same experimental conditions.
3
Histological Analysis of Biopsy Specimens
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Biopsy specimens were placed in 10% formalin saline for 24 hours and dehydrated by standard histological procedures. The biopsies were embedded in paraffin, cut using a microtome (5 μm sections), and mounted on glass slides. The slides were stained with haematoxylin and eosin, and sirius red staining were performed according to the manufacturer’s instructions (MUTO, Tokyo, Japan). For immunohistochemistry staining, mouse monoclonal antibodies directed against PCNA (Zhongshan, Beijing, China), Ki-67 (Zhongshan, Beijing, China), and PTB (Abcam, HK, China) were used and horseradish peroxidase-conjugated rabbit anti-mouse IgG (Abcam, HK, China) acted as the secondary antibody. Staining was achieved using a DAB Stain kit (Zhongshan, Beijing, China). The number of positive cells was counted in five random high power fields (400× total magnification).
4
Quantification of Recombinant Exendin-4 by ELISA
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The concentration of exendin-4 secreted by recombinant L. paracasei was measured by indirect ELISA according to the method described by Zhang et al. [41 (link)] with some modifications. Standard exendin-4 (Sigma, 0.2 μg/ml) was used as the coated antigen. Equal volumes (100 μl) of monoclonal antibody against exendin-4 (Abcam) (1:4000) and standard exendin-4 (0–100 nmol/l) or culture supernatant samples of L. paracasei L14/pMG76e-exendin-4, preincubated overnight at 4°C, were applied to detect the captured exendin-4. Horseradish peroxidase-conjugated rabbit anti-mouse IgG (Abcam) (100 μl) was used at a 1:10,000 dilution and revealed by reaction with TMBS substrate (Amresco). Absorption was determined at 450 nm and 630 nm by Multiskan MK3 ELISA plate reader (Thermo Labsystems). The exendin-4 concentration was calculated by the standard curve method.
5
Immunohistochemical Staining Protocol for CD3 and CD20
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Immunohistochemical staining was conducted as previously described (Bagabir et al., 2012 (link)). Briefly, antigen was retrieved by pressure cooking for 3 min in citrate buffer (pH = 6.0). Endogenous peroxidase was blocked in 0.3% hydrogen peroxide in methanol for 30 min, and nonspecific antibody binding was blocked with 1% bovine serum albumen (BSA). Mouse monoclonal antibodies directed against CD3 and CD20 (Zhongshan, Beijing, China) were used as primary antibodies and incubated overnight at 4°C. Horseradish peroxidase-conjugated rabbit anti-mouse IgG was incubated for 45 min at room temperature as the secondary antibody and was purchased from Abcam (Hong Kong, China). Staining was achieved using a diaminobenzidine (DAB) staining kit (Zhongshan, Beijing, China) at room temperature for 5 min. Sections were counterstained with hematoxylin.
6
Enzyme-Linked Immunosorbent Assay for Influenza Antibodies
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Sera were collected 2 wk after immunization to analyze antibody responses. Serum antibody titers against A-NP and B-NP were determined using ELISA. B/Yamagata/16/88 and A/PR/8 strains were split using 0.5% Triton X-100 (Sigma, St. Louis, MO, USA), and 96F Maxisorp immune plates were coated with the split viruses (3,600 PFU/well). The serum samples were diluted with 1% nonfat milk and 0.05% Tween 20/PBS and incubated at 25°C for 2 h. Thereafter, horseradish peroxidase-conjugated rabbit anti-mouse IgG (Abcam, Cambridge, UK) was added as a secondary antibody and incubated at 25°C for 1 h. Subsequently, 3,3′,5,5′-tetramethylbenzidine substrate and solution (KPL, Gaithersburg, MD, USA) were added to each well; thereafter, the reaction was terminated using 1 M phosphoric acid and detected at 450 nm using a Thermo Multiskan EX plate reader (Thermo Fisher Scientific, Vantaa, Finland).
ELISA results were expressed as endpoint titers. An endpoint titer is defined as the reciprocal of the highest analyte dilution that provides a reading above the cutoff. The cutoff formula used was as follows: “mean + 3 standard deviations of negative controls” (29 (link)).
ELISA results were expressed as endpoint titers. An endpoint titer is defined as the reciprocal of the highest analyte dilution that provides a reading above the cutoff. The cutoff formula used was as follows: “mean + 3 standard deviations of negative controls” (29 (link)).
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