Goat anti mouse immunoglobulin g

The 3-day-old neonatal mice were i.m. inoculated with 100 LD₅₀ of CVA4 YT226R strain (10^4.0 TCID₅₀/mouse) and mice with a clinical score of ≥4 were anaesthetized with ether and sacrificed. The lungs, hind limb muscles, spinal muscles and heart were separated, fixed, dehydrated, permeabilized and embedded in paraffin, which was then sliced into 4 μm sections. After staining with haematoxylin and eosin, histopathological analysis was performed.
Immunohistochemistry was performed using an avidin–biotin–immunoperoxidase technique. Briefly, tissue sections were dewaxed, dehydrated, and microwaved for 20 min at 99°C in a citrate buffer. Polyclonal mouse anti-CVA10 antibody (1:100 dilution; Genecreate, Wuhan, China) was applied for 2 h at 37°C. A secondary biotinylated goat anti-mouse immunoglobulin G (1:1000 dilution; Beyotime, Shanghai, China) antibody was added, followed by avidin–biotin–peroxidase complex and 3,3′-diaminobenzidine tetrahydrochloride chromogen. Tissues were counterstained with haematoxylin.

A549 cells were washed twice with PBS and lysed with 100 μL of immunoprecipitation buffer (Beyotime, Shanghai, China) containing 1% phenylmethanesulfonyl fluoride protease inhibitor (Beyotime). The proteins were extracted and resolved using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbit anti-FLAG (Abcam, Cambridge, UK), mouse anti-mCherry (Abcam) and mouse anti-β-actin (Beyotime) were used for immunoblotting analysis. β-actin-1 was used as the reference. Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse immunoglobulin G (Beyotime) were used as secondary antibodies.