To study microglial activation, we used monoclonal anti-ionized calcium-binding adapter molecule-1 (Iba-1) rabbit antibody (#019–19741, Wako Pure Chemical Industries, Ltd., Japan). Tissue sections were incubated with the primary antibody overnight at 4°C. A biotinylated universal secondary antibody was then applied for 1 h (anti-rabbit IgG, Vector Labs, Inc., Burlingame, CA), followed by an avidin biotin complex according to the manufacturer’s instructions (Vectastain Universal Elite Kit, Vector Labs). We visualized the antibody binding using the 3,3’-diaminobenzidine (DAB) peroxidase substrate kit (Vector Labs). We counterstained with hematoxylin and examined the sections using light microscopy on a Leica DMRB microscope (Leica Microsystems, Bannockburn, IL).
Vectastain universal elite kit
Manufactured by Vector Laboratories
The Vectastain Universal Elite Kit is a comprehensive immunohistochemistry reagent system designed for the detection of a wide range of primary antibodies. The kit includes all the necessary components, such as a biotinylated secondary antibody and an avidin-biotin enzyme complex, to perform sensitive and reliable immunohistochemical staining.
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2 protocols using vectastain universal elite kit
1
Microglial Activation Analysis via Iba-1 Immunohistochemistry
2
Immunohistochemical Analysis of GRK5 in Human Synovium
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Immunohistochemistry was performed on all human synovium tissue sections. Synovium samples were fixed in 4% paraformaldehyde for 2 days, delipidised, cut into sections that were 4 μm thick, and embedded in paraffin. Antigen retrieval was performed overnight with 1 mM EDTA at pH 8.0. Endogenous peroxidase activity was blocked with 3% hydrogen peroxidase in methanol for 30 min. For the blocking procedure, each specimen was placed in normal horse serum (VECTASTAIN Universal Elite Kit; Vector Laboratories, Burlingame, CA) for 20 min and then incubated for 1 h at room temperature with primary anti-GRK5 antibody (Proteintech, Rosemont, IL). Finally, the samples were counterstained with haematoxylin.
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