Six micrometer-thick sections were stained for the determination of total macrophages (goat αCD68; Santa Cruz Biotechnology, Santa Cruz, CA, USA). M2 macrophages were determined by staining for YM1 (goat αYM1/Chitinase 3-like 3; R&D Systems, Oxon, UK) and M1 macrophages for IRF-5 (rabbit αIRF-5; Santa Cruz Biotechnology) using the standard immunohistochemical procedure. Immunoreactivity was detected by sequential incubations of the lung section with a biotinylated secondary antibody, followed by peroxidase reagent (Vector Lab, Burlingame, CA, USA) and diaminobenzidine chromogen (Invitrogen, Carlsbad, CA, USA). The average stained area was quantified on the 8 to 10 images of mouse (n = 3–8) utilizing Image J software (National Institutes of Health, Bethesda, MD, USA). The number of cells was expressed per mm2 of tissue.
Diaminobenzidine chromogen
Manufactured by Thermo Fisher Scientific
Sourced in United States
Diaminobenzidine chromogen is a laboratory reagent used in various immunohistochemistry and histological staining techniques. It is a chromogenic substrate that produces a brown color reaction when used in conjunction with an enzyme label, such as horseradish peroxidase (HRP). The core function of diaminobenzidine chromogen is to facilitate the visualization of target molecules or cellular components in tissue sections or cell samples.
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3 protocols using diaminobenzidine chromogen
1
Macrophage Subtype Quantification in Lung Tissue
2
Quantifying Lung Collagen and α-SMA
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Six-micrometre-thick sections of lung tissue from each paraffin block were deparaffinized with xylene and hydrated in ethanol. For immunohistochemical detection of collagen III, collagen V, and α-SMA, the lung sections were incubated overnight at 4°C with anti-collagen III antibody, anti-collagen V antibody (both from Abcam, Cambridge, MA, USA), and anti-α-SMA antibody (Dako, High Wycombe, UK). Immunoreactivity was detected by sequential incubation of lung sections with a biotinylated secondary antibody followed by peroxidase reagent (Vector Laboratories, Burlingame, CA, USA) and diaminobenzidine chromogen (Invitrogen, Carlsbad, CA, USA). The results are expressed as the area of immunostaining per micrometre length of the basement membrane of bronchioles with an internal diameter of 150 to 200 μm. At least 10 bronchioles were counted in each slide using a slide scanner (Pannoramic MIDI, 3DHISTECH Ltd., Budapest, Hungary).
3
Quantification of α-SMA in Lung Tissue
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Alpha-smooth muscle actin (α-SMA) was immunohistochemically detected as described previously23 (link). Briefly, paraffin blocks were sectioned at a thickness of 6 μm, deparaffinized in xylene, rehydrated in ethanol, and incubated overnight at 4ºC with a primary monoclonal antibody against α-SMA (titer 1:50; Dako, High Wycombe, UK). Immunoreactivity was assessed by further incubation with a biotinylated secondary antibody, followed by peroxidase treatment (Vector Laboratories, Burlingame, CA, USA) and the addition of a diaminobenzidine chromogen (Invitrogen, Carlsbad, CA, USA). The α-SMA-immunostained area in each lung section was outlined and quantified using a light microscope and image analysis system (BX50; Olympus, Tokyo, Japan). The immunostained areas of the bronchiolar basement membranes (internal diameter = 150–200 μm) were derived. At least 10 bronchioles were counted in each slide.
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