At day 5 p.i., mice were sacrificed and lung tissues were harvested. Paraformaldehyde-fixed paraffinized lung sections (4-μm) were deparaffinized and dehydrated. Lung sections were stained with hematoxylin and eosin for histopathology analysis. For immumohistochemistry, lung sections were microwaved for 20 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval. Hydrogen peroxide (H2O2; 3%) was used to block peroxidase activity. Sections were blocked with 5% BSA in TBST for 1 h at room temperature and incubated with rabbit anti-mouse CD3 antibody in the humidified chamber for overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:200; Multi-Sciences). Sections were developed with DAB reagent (Maixin, China) and counterstained with hematoxylin.
Horseradish peroxidase hrp conjugated secondary antibody
Manufactured by MultiSciences Biotech
Sourced in China
Horseradish peroxidase (HRP)-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques. It is a secondary antibody that has been conjugated with the enzyme horseradish peroxidase. The HRP enzyme can catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and visualization of target antigens in samples.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dab reagent, by Maixin Group (1 mentions)
Acridine orange, by Avantor (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Beyotime (1 mentions)
Ecl plus kit, by Beyotime (1 mentions)
Proteinase k, by Beyotime (1 mentions)
Ecl reagent kit, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Takara Bio (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ecl reagent, by PerkinElmer (1 mentions)
0.2 μm pvdf membrane, by Bio-Rad (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Endostatin, by Abcam (1 mentions)
Vascular endothelial growth factor (vegf), by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by MultiSciences Biotech (1 mentions)
6 protocols using horseradish peroxidase hrp conjugated secondary antibody
1
Immunohistochemical Analysis of Lung Inflammation
2
Comprehensive Cell Viability Assay
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RPMI-1640 medium (Gibco, USA), fetal bovine serum(FBS, Gibco, USA), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Fluka, USA), acridine orange (AO, Amresco, USA), ethidium bromide (EB, Sigma, USA), Wright-Giemsa dye solution (Nanjing Jiangcheng Bioengineering Institute, Nanjing, China), RNase A (Fermantas, Canada), proteinase K (Fermantas, Canada), ECL plus kit (Beyotime Institute of Biotechnology, China), DL 2000 DNA Marker (Takata, Japan), prestained protein marker (fermentas, Thermo Scientific, USA), antibody to GAPDH, GRP78, GADD153, Bcl-2, Bax, p53, survivin, Actived-Caspase 3 p17 (Bioworld Technology, MN, USA), horseradish peroxidase (HRP)-conjugated secondary antibody (MultiSciences, China). All other biochemicals and chemicals used in the experiment were of analytical grade.
3
Protein Expression Analysis via Western Blotting
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Cells or tumor tissues were lysed with RIPA buffer (Takara Bio, Japan) for extracting total protein. Protein concentration was quantified using BCA Protein Assay Kit (Thermo Fisher Scientific, CA, USA). Proteins were separated using 10% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes. Next, membranes were incubated with blocking buffer for 1 h, followed by the incubation with primary antibody against CBX7, ERK1/2, p-ERK1/2, p38, p-p38, and GAPDH (1 : 1,000, Abcam, UK) at 4°C overnight. After being washed with 1× TBST buffer for three times, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1 : 500, MultiSciences, China) for 2 h at room temperature in the dark. The protein bands were visualized using an ECL reagent kit (Thermo Fisher Scientific, CA, USA) and observed under a ChemiDoc™ imaging system (Bio-Rad, CA, USA). The relative protein expression was calculated via normalizing to GAPDH.
4
Protein Quantification and Western Blot Analysis
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Cells and skin tissue were collected and lysed as previously reported [29] (link), [30] (link), [31] (link). The protein concentration was quantified using a BCA kit (Beyotime, China). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel and transferred to a polyvinylidene difluoride membrane. The membranes were blocked using 5% skim milk for 1 h, and then incubated with primary antibodies at 4 °C overnight. After proper washing, the membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (MULTI SCIENCES, China) for 1 h. At last, proteins were detected using an ECL kit and an automatic chemiluminescence image analysis system. Quantitative analysis was performed using Image J software.
5
Western Blot Analysis of Protein Extracts
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Excised lungs and cells were lysed using ice-cold RIPA buffer (25 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) supplemented with protease inhibitors (Sigma). The crude lysates were centrifuged at 14,000 rpm (13,000 × g) for 15 min at 4 °C, and the supernatant was collected. Equal amounts of cell or tissue extracts were loaded onto 10% SDS-polyacrylamide gels for separation. Then, proteins were transferred to 0.2 μm PVDF membranes (Bio-Rad), which allowed subsequent probing with primary antibodies. After overnight incubation at 4 °C, the membranes were washed with 0.1% TBST (TBS/0.1% Tween 20) and incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Multisciences). The signals were visualized using enhanced chemiluminescence (ECL) reagents (Perkin-Elmer). Quantification of the relative band intensities was performed using ImageJ software version 1.43.
6
Western Blot Analysis of Angiogenic Factors
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Whole cells were lysed and protein samples containing 40 μg of protein were separated on 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), then electro-transferred onto polyvinylidene fluoride (PVDF) membranes (MilliporeCorp., Bedford, MA, USA). They were then blocked in TRIS-buffered saline containing Tween 20 (TBST; China National Pharmaceutical Group Corporation; Beijing, China) and 5% nonfat milk for 2 h to block nonspecific binding, then rinsed with TBST. The blots were incubated with primary first antibodies to endostatin (1:1000; Abcam, UK), VEGF (1:1,000; Proteintech, USA), and GAPDH (1:2000; MultiSciences, Hangzhou, China) at 4°C overnight. After the membranes were washed several times with TBST, appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:20,000; MultiSciences, Hangzhou, China) were added for 2 h at room temperature. Finally, the membranes were washed, and the enhanced chemiluminescence (ECL) substrates were detected (GE Healthcare, USA). GAPDH was used as a loading control. Data were analyzed with Gel-Pro Analyzer software.
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