To further confirm that NS1 induced CD138 shedding in endothelial cells in mice, 50 μg of recombinant NS1, E or prM protein or 50 μl of PBS was subcutaneously injected into 8- to 12-week-old BALB/c mice, followed by a second injection of an equal amount of recombinant proteins or PBS 24 h after the first injection at the same site. The mice were sacrificed 24 h after the second injection. The separated skin tissues were fixed in formalin overnight and embedded in paraffin for the preparation of a series of sections. After paraffin removal and antigen retrieval by citrate buffer, the tissue sections were blocked, and immunohistochemistry was performed using the Mouse/Rabbit HRP Detection System with DAB (brown) (BioTnA Biotech, Kaohsiung, Taiwan). Hematoxylin was used as a counterstain. Anti-α-SMA antibody (Arigo, Hsinchu City, Taiwan) was used at 1:200, and anti-CD138 antibody (BD, Franklin Lakes, NJ) was used at 1:100. The resultant images were acquired using phase-contrast microscopy (Olympus, Tokyo, Japan).
Anti α sma antibody
Manufactured by Arigo Biolaboratories
The Anti-α-SMA antibody is a laboratory reagent used to detect the presence of alpha-smooth muscle actin (α-SMA) in biological samples. α-SMA is a cytoskeletal protein found in vascular smooth muscle cells and is commonly used as a marker for myofibroblasts and activated stellate cells. This antibody can be used in various immunoassay techniques, such as immunohistochemistry and Western blotting, to visualize and quantify α-SMA expression in research applications.
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2 protocols using anti α sma antibody
1
Subcutaneous NS1 Injection Induces CD138 Shedding in Mice
2
Immunofluorescence Staining of Vascular Smooth Muscle Cells and Myofibroblasts
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Immunofluorescence staining against α-SMA was performed to detect vascular smooth muscle cells (26 (link)) and myofibroblasts (27 (link)), according to the manufacturer’s protocol. Briefly, after dewaxing, rehydration, and antigen retrieval, the sections were blocked with 2% bovine serum albumin in phosphate buffer saline (PBS) for 1 h. Sections were incubated with anti-α-SMA antibody (Arigo Biolaboratories, Taiwan, China) overnight at 4 °C and then with goat anti-mouse secondary antibody labeled with Alexa 488 (Molecular Probes, Invitrogen, Carlsbad, CA) for 1 h at 37 °C. At the end of the incubation period, the sections were washed with PBS and counterstained with DAPI (Life Technologies, CA, USA). Images were observed using fluorescence microscopy (IX53, Olympus) for further semiquantitative analysis. For each section of the intraosseous or intra-articular part, six random fields of view (FOV) around the graft were randomly selected, and α-SMA positive vessels and myofibroblasts were counted by two independent observers using Image-Pro Plus 6.0. (27 (link),28 (link)). The positive ellipse was an α-SMA-positive vessel, and the positive spindle cell was an α-SMA-positive myofibroblast.
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