Tissues were collected, sectioned into a thickness of 8 μm, dewaxed in xylene and rehydrated in graded ethanol. Subsequently, tissues were soaked in 3% H2O2 for 20 min to block endogenous peroxidase. Tissues were incubated with anti-CX3CR1 (cat. no. ab95620; 1:2,000) or anti-CX3CL1 antibody (cat. no. ab89229; 1:2,000) (from Abcam) at 4°C overnight. Following secondary antibodies (cat. nos. ab150077 and ab150115; 1:2,000; Abcam) incubation, immunoperoxidase staining was performed using the VECTASTAIN Elite ABC kit (cat. no. AK-5001; Vector Laboratories, Inc.) and 3,3′-diaminobidine-tetrachloric acid as a chromogenic agent. The sections were observed using an optical microscope (Olympus-IX51; Olympus Corporation).
Vectastain elite abc kit
Manufactured by Abcam
The VECTASTAIN Elite ABC kit is a laboratory reagent used for the detection and visualization of target antigens in biological samples. It utilizes an avidin-biotin-peroxidase complex (ABC) system to amplify the signal, enhancing the sensitivity of the detection process. The kit provides the necessary components to perform immunohistochemical or immunocytochemical staining procedures.
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2 protocols using vectastain elite abc kit
1
Immunohistochemical Analysis of CX3CR1 and CX3CL1
2
Immunohistochemical Analysis of Lung Tissue
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Lung samples were fixed in formaldehyde, embedded in paraffin and serial sections were prepared for cell (alpha-smooth muscle actin-α-sma cells) and ECM glycoprotein staining. Tissue sections were deparaffinized, rehydrated and then antigen was retrieved by being boiled in Tris-EDTA buffer (pH 9.0) for 5 min. Samples were blocked with 0,3% H2O2 for 1 h with avidin/biotin blocking kit (vector). Immunohistochemistry was carried out using primary antibody of tenascin C (1/300, Abcam), versican (1/250, Lifespan) and fibronectin domain EDA (1/400, Abcam), followed by biotin-avidin/peroxidase (Vectastain Elite ABC kit) incubation. Alpha-smooth muscle actin (α-sma) (RTU Flex, Dako) was performed with Autostainer Link Instrument, using EnVision Flex+, mouse, and high pH (Link) (Dako), according to the manufacturer’s recommendation. All sections were counterstained with haematoxylin.
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