EC patient tissue samples were obtained, fixed in 4% paraformaldehyde, and embedded into paraffin for sectioning with a microtome. The specimens were incubated with primary antibodies against CXCR4 (1:500, Abcam, UK), CD133 (1:100, Novus Biologicals USA) over night. The sections were treated with biotinylated anti-rabbit IgG antibody (Nichirei biosciences, Japan). Color developing agent was obtained by treatment with DAB kit (Nichirei biosciences, Japan). The samples were examined using a Leica microscope (Leica DMRBE, Germany).
Dab kit
Manufactured by Nichirei Biosciences
Sourced in Japan
The DAB kit is a laboratory reagent used to detect the presence of a specific protein or molecule in a sample. It functions by producing a colored reaction that indicates the location and relative abundance of the target analyte within the sample.
Automatically generated - may contain errors
Lab products found in correlation
Cxcr4, by Abcam (1 mentions)
Cd133, by Novus Biologicals (1 mentions)
Protein block, by Agilent Technologies (1 mentions)
Epr19025 42, by Abcam (1 mentions)
Smear gell, by Genostaff (1 mentions)
Iba 1, by Abcam (1 mentions)
Dmrbe microscope, by Leica (1 mentions)
Alpha smooth muscle actin α sma, by Abcam (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
Tgf β1, by Abcam (1 mentions)
Cd163, by Abcam (1 mentions)
3 protocols using dab kit
1
Immunohistochemical analysis of CXCR4 and CD133
2
Immunohistochemical Analysis of MSLN Expression
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MSLN protein expression was examined by immunohistochemical staining in MSLN cDNA-transduced iPSDCs and ubiquitin-MSLN combined cDNA-transduced iPSDCs. iPSDCs were collected using Smear Gell (GenoStaff, Tokyo, Japan). The endogenous peroxidase activity was suppressed by a solution of 0.3% hydrogen peroxide in methanol for 30 min. After being rinsed in Tris-buffered saline (TBS), the sections were incubated with a blocking reagent; Protein block (Dako, Kyoto, Japan) for 20 min at room temperature. The sections were incubated for 30 min at room temperature with the primary antibody, a 1:1500 dilution of an anti-human MSLN monoclonal antibody (EPR19025-42, Abcam, Cambridge, UK). After rinsing in TBS, the primary antibody was visualized using anti-rabbit antibody (Dako) for 60 min at room temperature, and DAB kit (Nichirei, Tokyo, Japan).
3
Histological Analysis of Skin Wound Healing
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All tissues newly formed at the full-thickness skin excision site on PODs 1, 4 and 7 were collected from each mouse. These resected samples were dehydrated in a series of graded ethanol baths and a xylene bath, prior to being immersed in paraffin wax. The samples were then embedded in paraffin molds and sliced into 4 μm-thick sections that were mounted on glass slides and stained with hematoxylin and eosin (H&E) and azan. Immunohistochemical staining was performed for the specimens on PODs 1, 4 and 7. The specimens were incubated overnight with a primary antibody against either CD163 (1:500; Abcam, Cambridge, UK), TGF-β1 (20 μg/ml, Abcam), VEGF (10 μg/ml, Abcam), CD31 (1:500, Abcam), alpha-smooth muscle actin (α-SMA) (1:800, 4 μg/ml, Abcam) or Iba1 (1:200, 1.5 μg/ml, Abcam). The sections were treated with biotinylated anti-rabbit, anti-mouse or anti-goat IgG secondary antibody (Nichirei Biosciences Inc., Tokyo, Japan). A color-developing agent was obtained by treatment with a DAB kit (Nichirei Biosciences Inc.). The stained tissues were examined using a Leica DMRBE microscope (Leica, Wetzlar, Germany) and digital camera DP73 (Olympus Co.).
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