Mouse neutrophils were isolated from mouse bone marrow using EasySep mouse neutrophil enrichment kit and EasySep Magnet according to manufacturer’s instructions (StemCell). These neutrophils (5×105 cells per well) were then treated with CGRP (0.01–1 µM, GenScript) or vehicle (PBS), immediately prior to mixture with S. pyogenes (5×103 cfu) M1 wt strain or vehicle (PBS) in 200 µL of neurobasal-A medium containing 10% of fresh mouse serum. Serum was obtained after coagulation of whole mouse blood. Plates were incubated for 30 min at 37°C with gently shaking (150 rpm), and supernatant collected for MPO activity measurements. Supernatants (50 µL) were added to 200 µL of 50 mM phosphate buffer solution (pH 6.0) containing 0.167 mg/mL of peroxidase substrate o-dianisidine dihydrochloride (Santa Cruz Biotech) and 0.05% hydrogen peroxide (Santa Cruz Biotech) and incubated for 30 min at room temperature. MPO activity was determined spectrophotometrically by measuring the increase in absorbance at 450 nm.
Hydrogen peroxide
Manufactured by Santa Cruz Biotechnology
Sourced in United States
0.3% hydrogen peroxide is a chemical solution commonly used in laboratory settings. It serves as a mild oxidizing agent and disinfectant.
Automatically generated - may contain errors
Lab products found in correlation
Acrymount, by StatLab (5 mentions)
Hrp dab system, by Merck Group (4 mentions)
Triton x 100, by Merck Group (2 mentions)
Ab260040, by Merck Group (2 mentions)
Eclipse ni u, by Nikon (2 mentions)
Horseradish peroxidase 3 3 diaminobenzidine system, by Merck Group (2 mentions)
Lionheart fx, by Agilent Technologies (2 mentions)
Ab260040, by Abcam (2 mentions)
Easysep magnet, by STEMCELL (1 mentions)
Easysep mouse neutrophil enrichment kit, by STEMCELL (1 mentions)
Bovine serum albumin (bsa), by MP Biomedicals (1 mentions)
Sc 1350, by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
4 phenylbutyric acid 4 pba, by Santa Cruz Biotechnology (1 mentions)
Image pro plus software 6, by Media Cybernetics (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
12 protocols using hydrogen peroxide
1
Neutrophil Response to S. pyogenes
2
Immunohistochemical Detection of TNF-α in Lung Tissue
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Paraffin sections fixed on gelatin-coated slides were deparaffinized and rehydrated prior to sequential incubation with 0.3% Triton X-100 (Sigma-Aldrich), 3% hydrogen peroxide (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and 2% bovine serum albumin (MP Biomedicals, Santa Ana, CA, USA). The sections were subsequently incubated overnight at 4°C with a goat polyclonal primary antibody against TNF-α (1:400; sc-1350; Santa Cruz Biotechnology Inc.). The following day, the lung tissue was incubated with a biotinylated rabbit anti-goat secondary antibody (Wuhan Boster Biotechnology Co., Ltd., Wuhan, China) for 30 min, prior to immunoreactivity detection using a 3-amino-9-ethylcarbazole peroxidase substrate kit (Wuhan Boster Biotechnology Co., Ltd.).
3
Induction of ER and Oxidative Stress in INS-1E Cells
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The INS-1E cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 11 mM glucose supplemented with 10 mM HEPES (pH 7.3), 10% heat-inactivated fetal bovine serum (FBS; Invitrogen), 50 μM β-mercaptoethanol, 1 mM sodium pyruvate, 50 μg/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO2 in a humidified incubator. ER stresses are induced by thapsigargin (1 μM; Calbiochem, San Diego, MO, USA) for 6 h or tunicamycin (2 μg/mL; Calbiochem) for 16 h treatments with/without 4-phenylbutyric acid (4-PBA, 20 μM; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Oxidative stresses are induced by serum deprivation, nitric oxide (2 mM; Santa Cruz Biotechnology), or hydrogen peroxide (200 μM; Santa Cruz Biotechnology) for 24 h treatments.
4
Endothelial Cell Staining for Angiogenesis
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Specific staining for endothelial cells was conducted using the neo-angiogenesis marker CD31. The slides were fixed using cold acetone (Cusabio Biotech Co., Ltd.) for 20 min. Following two washes with PBS, the tissue sections were incubated with 3% hydrogen peroxide (Santa Cruz Biotechnology, Inc.) in methanol for 30 min, in order to block endogeneous peroxidase activity. Primary antibody incubation was conducted at 37°C for 2 h, the slides were then incubated with goat anti-mouse IgG secondary antibody for 30 min, and with streptavidin biotin-peroxidase complex (Santa Cruz Biotechnology, Inc.) for 40 min. Following incubation with diaminobenzidine chromogen (Santa Cruz Biotechnology, Inc.), the tissue sections were re-stained with hematoxylin. Vessel density was determined by counting the number of microvessels per high-power field (Olympus IX 70; Olympus Corporation, Tokyo, Japan).
5
Immunohistochemical Analysis of TNF-α Expression
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Serial sections (5 µm) were fixed on gelatin-coated slides. Following deparaffinization with two changes of xylene, rehydration with graded ethanol and sequential incubation for 5 min at room temperature with 0.3% Triton X-100 (Sigma-Aldrich) and 3% hydrogen peroxide (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), the sections were incubated with goat polyclonal primary antibody against TNF-α (1:400 dilution; cat. no. sc-1350; Santa Cruz Biotechnology, Inc.) for 12 h at 4°C. Following three washes with PBS, the sections were incubated for 30 min at room temperature with biotinylated rabbit anti-goat monoclonal antibody (1:100 dilution; cat. no. BA-1006; Wuhan Boster Biological Technology, Ltd., Wuhan, China), and the immunoreactivity detected with a 3-amino-9-ethylcarbazole peroxidase substrate kit (Wuhan Boster Biological Technology, Ltd.). The sections were counterstained with hematoxylin, and observed under the Olympus BX53 microscope. Mean optical density (OD) was subsequently calculated using Image-Pro Plus Software 6.0 (Media Cybernetics, Rockville, MD, USA).
6
Immunofluorescence Localization of Nephrin
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Renal tissues were fixed using 4% paraformaldehyde (Santa Cruz, USA) to produce paraffin (Sigma-Aldrich, USA)-embedded sections that were analyzed using immunofluorescence. The sections were first dewaxed and dehydrated to recover antigens. Endogenous peroxidase activity was halted using 3% hydrogen peroxide (Santa Cruz, USA) (diluted with PBS). Next, 5% goat serum (Thermo Scientific, USA) (diluted with PBS) was used to block the sections for 30 minutes. The samples were then incubated overnight with the nephrin antibody (1 : 100) (diluted with 5% goat serum) at 4°C, followed by incubation with the secondary antibody (Invitrogen, USA) (diluted with 1% BSA) for an hour at 37°C. Finally, the sections were stained with DAPI (Santa Cruz, USA). The stained cell images were produced using a fluorescence microscope.
7
Quantifying Collagen X Expression in Growth Plate
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Expression of type X collagen (Col X) is an indicator of chondrocyte hypertrophy and marks the mineralization front of both the growth plate and enthesis.3 Immunohistochemistry was used to assess the distribution of Col X, marker of the mineralization front in the ECM. Sectioned slides from Young samples were deparaffinized and rehydrated to 70% ethanol (n > = 4/genotype). Heat‐mediated antigen retrieval was performed at 65°C (sodium citrate Buffer, pH 6.0). Slides were quenched and blocked at room temperature using 0.3% hydrogen peroxide (Santa Cruz, Dallas, TX) and 5% goat serum in PBS, respectively. Primary rabbit monoclonal anti‐collagen X antibody (Abcam, ab260040; 1:100) with HRP/DAB system (Millipore Sigma) was used for detection of Col X. Slides were counterstained with hematoxylin and cover‐slipped with acrylic mounting media (Acrymount, Statlab, McKinney, TX, USA). The presence of Col X in the ECM was quantified at the secondary ossification center by segmentation based on cellular and tissue morphology, and the region with Col X localization was measured using ImageJ software.26
8
Immunohistochemical Analysis of Collagen X
9
Histological Analysis of Loaded Mouse Hindlimbs
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Distal hindlimbs from mice treated with 20-min of loading for 3 weeks (19 days) as well as from naïve controls were decalcified in 14% EDTA and processed for paraffin embedding. Tissues were sectioned at 6 μm in the sagittal plane and stained with hematoxylin and eosin or Toluidine blue and coverslipped with an acrylic mounting media (Acrymount, StatLab, McKinnery, TX, USA) or used for immunohistochemistry. For immunohistochemistry, paraffin sections were deparaffinized and quenched in 0.3% hydrogen peroxide (Santa Cruz Biotechnology, Dallas, TX), followed by heat-mediated antigen retrieval and blocking (5% goat serum in phosphate buffer saline). Slides were then incubated in primary antibodies (listed in Table 1 ) overnight at 4°C, followed by washing and incubation with appropriate secondary antibodies. Horseradish peroxidase 3,3′-diaminobenzidine system (MilliporeSigma, Billerica, MA, USA) was used for detection, and slides were counterstained with hematoxylin and coverslipped with Acrymount (Acrymount, StatLab, McKinney, TX, USA). All slides were imaged on a bright-field microscope (ECLIPSE Ni-U, Nikon).
10
Collagen X Expression in Developing Mice
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