The eyes of 8 and 48-week-old hDCN-Tg and WT mice were fixed for 48 h in Super Fix KY-500 solution® (Kurabo, Tokyo, Japan), embedded in paraffin, and sectioned at approximately 4 µm (n = 4). Then, the eyes were stained with H & E. For immunohistochemical analysis, the eyes were immunostained using a tyramide signal amplification (TSATM) kit (Molecular Probes Inc., ThermoFisher Scientific Japan Ltd.), following the manufacturer’s protocol and as described previously [25 (link),26 (link)]. For DCN immunostaining, the sections were treated with target retrieval solution (Dako/Agilent, Santa Clara, CA, USA) following treatment with 0.2 U/mL protease-free C-ABC as described in the section on western blotting to expose the core protein before blocking with a blocking reagent (Molecular Probes Inc., ThermoFisher Scientific Japan Ltd.). DCN was visualized using anti-rabbit DCN polyclonal antibody (ab137508; abcam). The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Fluoroshield Mounting Medium with DAPI: ImmunoBioScience Corp., Mukilteo, WA, USA). For negative controls (NC), rabbit and mouse IgG isotype controls (Dako) were used, and the primary antibody was omitted. NC was performed along with the experiments.
Ab137508
Manufactured by Abcam
Ab137508 is a lab equipment product. It is a core component that serves a specific function in laboratory workflows.
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4 protocols using ab137508
1
Immunohistochemical Analysis of DCN in Transgenic Mice
2
Temporal Analysis of Wound Healing
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Skin from the excisional model was harvested at 3, 7, 21, and 30 days following wounding. These wound biopsies were fixed in 10% buffered formalin, processed, and embedded in paraffin blocks using standard protocols [23 (link)]. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) and analyzed for general tissue and cellular morphology. Masson’s trichrome and Picrosirius Red staining using MetaMorph software analysis evaluated collagen deposits and collagen alignment [24 ]. Sections were stained using immunohistochemistry for immunohistochemically for tenascin-C (1:100, ab137508; Abcam), fibronectin (1:100, ab2413; Abcam), and CD3 (1:250, ab5690; Abcam).
3
Immunofluorescence Staining of Cells
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After the cells grew on the glass slide, they were fixed with pre-cooled paraformaldehyde, washed with PBS, permeated with 0.2% Triton X-100 for 10 min, and washed again with PBS for three times; then cells were sealed with the serum of the same host for 30 min, and washed with PBS for three times. The primary antibodies with cat. nos. ab150301 and ab137508 (Abcam) were added in the wet box overnight, and then cells were washed with PBS for three times, the secondary antibody with cat. no. ab6789 (Abcam) was added at room temperature for 2 h in a dark place, and washed again with PBS for three times. After nuclear staining with DAPI, they were photographed under fluorescence microscope (SZ61; Olympus Corporation, Tokyo, Japan).
4
Western Blot Analysis of Protein Expression
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The fibroblasts in experimental and control groups were collected, and the cells were resuspended using the cell lysis buffer, and cleaved on ice for 30 min, followed by centrifugation at 12,000 × g at 4°C for 15 min. The supernatant was carefully absorbed as the total protein. After protein quantification using the BAC protein quantification kit, the loading buffer was added, 60 µg proteins in each group were taken for 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was sealed using Tris-buffered saline (TBS) containing 5% skimmed milk at room temperature for 1 h, primary antibodies with cat. nos. ab150301 and ab137508 both from Abcam, were added dropwise for incubation at 4°C overnight. On the next day, the membrane was washed with Tris-buffered saline Tween (TBST) for three times (15 min/time). Then secondary antibody, goat anti-mouse IgG-HRP (1:2,000; cat. no. ab6789; Abcam) was added for incubation at room temperature for 1 h, followed by washing with TBST, luminous reaction using electrochemiluminescence (ECL) kit, image scanning and analysis, and gray scale analysis with glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the internal reference.
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