Section (6-µm thickness) from paraffin-embedded mouse skin and lungs were deparaffinized and incubated overnight at 4 °C with mouse mAbs to CD3 (1:200; Nichirei Bioscience), F4/80 (1:1600; Abcam), and α-SMA (1:200; DAKO), followed by incubation with peroxidase-labeled secondary antibody (Nichirei BioScience) and color development with the aminoethyl carbazole system (Nichirei BioScience) [28 (link)]. Immunostained cells were counted in three high-power microscopic fields. Each section was examined independently by two investigators (T.C. and N.O.) in a blinded manner.
Peroxidase labeled secondary antibody
Manufactured by Nichirei Biosciences
Sourced in Japan
Peroxidase-labeled secondary antibody is a commonly used reagent in immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and Western blotting. It consists of a secondary antibody that is conjugated with the enzyme horseradish peroxidase. This labeling enables the detection and quantification of target antigens or proteins in biological samples through a colorimetric or chemiluminescent signal.
Automatically generated - may contain errors
Lab products found in correlation
F4 80, by Abcam (3 mentions)
Aminoethyl carbazole system, by Nichirei Biosciences (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
α sma, by Agilent Technologies (1 mentions)
P smad3, by Santa Cruz Biotechnology (1 mentions)
Image lab 2, by Bio-Rad (1 mentions)
Rabbit anti hif 1α, by Cell Signaling Technology (1 mentions)
Rabbit anti vegf, by Santa Cruz Biotechnology (1 mentions)
Gradient acrylamide gel, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Polyvinylidene fluoride microporous membrane, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Blocking reagent, by Toyobo (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Ponceau s, by Nacalai Tesque (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Anti p smad3 antibody, by Cell Signaling Technology (1 mentions)
6 protocols using peroxidase labeled secondary antibody
1
Immunohistochemistry of Mouse Skin and Lungs
2
Immunohistochemical Analysis of Skin
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Sections (6 μm in thickness) from paraffin-embedded mouse skin were incubated for 120 minutes at room temperature with monoclonal antibodies (mAbs) to CD3 (1:200; Nichirei Biosciences, Tokyo, Japan), F4/80 (1:1600; Abcam), and p-Smad3 (1:50; Santa Cruz Biotechnology), then with peroxidase-labeled secondary antibody (Nichirei Biosciences), followed by color development with the aminoethylcarbazole system (Nichirei Biosciences). CD3+ cells, F4/80+ cells, and p-Smad3-positive cells were counted under a high-power microscopic field (the Hall section for CD3+ cells and distinct fields for the F4/80+ cells and p-Smad3-positive cells). Each section was examined independently by two investigators (TC and NO) in a blinded manner.
3
Western Blot Analysis of Mesenchymal Stem Cells
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Protein samples for western blot analysis were prepared as described previously [22] (link). Briefly, MSCs (passages 3–5) were washed three times with ice-cold PBS and then treated with lysis buffer (50 mM Tris-HCl, pH 7.5, containing 2% SDS (Sigma-Aldrich) and a protease inhibitor cocktail (Roche, Mannheim, Germany). Samples were centrifuged for 1 h at 18,000× g at 4°C. The supernatants were collected as whole cell lysates. Protein concentrations were estimated using a DC protein assay (Bio-Rad) with a bovine serum albumin standard. Equal amounts of proteins (10 µg) were resolved by SDS-polyacrylamide gel electrophoresis on 4–20% acrylamide gradient gels (Bio-Rad) and then transferred onto a polyvinylidene fluoride microporous membrane (Millipore, Billerica, MA). The membranes were blocked with a blocking reagent (Toyobo, Tokyo, Japan) and then incubated with each primary antibody. The primary antibodies used were: rabbit anti-PCAF, rabbit anti-HIF-1α (Cell Signaling Technology), rabbit anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-β-actin (Cell Signaling Technology). After washing, the membranes were incubated with a peroxidase-labeled secondary antibody (Nichirei, Tokyo, Japan) and visualized using Immunostar LD (Wako). Images were captured digitally using a ChemiDoc XRS+ (Bio-Rad) and analyzed by Image Lab 2.0.1 software (Bio-Rad).
4
Quantitative Western Blot Analysis of Cerebral Proteins
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Western blot analysis was performed according to a previously described procedure with minor modifications.49 (link) Cerebral tissues were homogenized in 9 volumes of 50 mM Tris-HCl, pH 7.5 (Tris buffer), and protease inhibitor cocktail (PIC; Roche Diagnostics, Indianapolis, IN), followed by sonication and centrifugation at 18,000 × g for 1 h at 4°C. Residual pellets were lysed with 7 volumes of Tris buffer containing 2% sodium dodecyl sulfate (SDS) and PIC, followed by sonication and centrifugation. Samples containing equal amounts of proteins were resolved by SDS-PAGE on 4%–20% TGX (Tris-glycine extended) gels and transferred onto nitrocellulose membranes using the Trans-Blot Turbo System (Bio-Rad, Hercules, CA). The membranes were stained with Ponceau S (Nacalai, Tokyo) for the analysis of total proteins. After a brief wash, the membranes were dipped in a blocking buffer (Tris buffer containing 150 mM NaCl, 0.1% gelatin, 0.1% casein, and 0.05% Tween 20) and incubated with each primary antibody. Membranes were then immunostained with peroxidase-labeled secondary antibody (Nichirei, Tokyo, Japan) and analyzed using a Chemi Doc XRS + System and Image Lab software (Bio-Rad). Quantitative analysis of the target band was performed by normalizing the total protein.
5
Immunohistochemical Analysis of Mouse Skin and Lung
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Briefly, the mouse skin and lung sections were deparaffinized, and antigen-retrieved by incubating with HistoVT One (Nacalai Tesque) at 90°C for 10 min. Subsequently, the slides were incubated for 10 min with solution to block endogenous peroxidase and alkaline phosphatase (BLOXALL, Vector Laboratories). Next, the slides were immunostained with monoclonal antibodies against mouse CD3 (Adjusted, Nichirei Biosciences) or F4/80 (1:250, Abcam), anti-αSMA antibody (1:200, Abcam), anti-p-Smad3 antibody (1:200, Cell Signaling Technology, USA), anti-calpain 1 antibody (1:200, Abcam), anti-calpain 2 antibody (1:200, Abcam), peroxidase-labeled secondary antibody (Nichirei Biosciences), and a coloration substrate system with 3,3-diaminobenzidine (DAB, Nichirei Biosciences). αSMA- or Calpain-positive area was assessed as follows. One representative section of each mouse was photographed at five random locations in the 200 × field of the microscopic view for analysis. Then, the αSMA- or calpain-positive area was determined through pixelization of the staining area using Photoshop software (ver. 16).
6
Immunohistochemical Analysis of NOX1 and NOX4 in Achilles Tendons
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Four Achilles tendons of rats in each group were used for immunohistological analysis of NOX1 and NOX4. Longitudinal sections of frozen Achilles tendons were cut to a thickness of 7 μm and fixed with 10% phosphate-buffered paraformaldehyde at room temperature. The sections were incubated with proteinase for 10 min and treated with 3% hydrogen peroxide (Wako Pure Chemical Corp.) to block endogenous peroxidase activity, and then incubated with anti-NOX1 or anti-NOX4 antibodies (both 1:1000; Abcam, Cambridge, UK) overnight at 4 °C. Sections were incubated with a peroxidase-labeled secondary antibody (Nichirei Bioscience, Tokyo, Japan) for 30 min at room temperature, followed by incubation with 3,3′-diaminobenzidine peroxidase substrate (Nichirei Bioscience) [11 (link), 27 (link)]. Sections were counterstained with hematoxylin. To quantitatively evaluate NOX1 and NOX4 immunostaining, the percentage of NOX1 and NOX4 staining was calculated by two independent researchers using Adobe Photoshop CC 2020 software (Adobe Systems Inc., San Jose, CA, USA) as the ratio of stained brown pixels to total pixels of the entire tendon in a randomly selected area from each tendon slide.
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