Goat anti rabbit igg hrp conjugated antibody

Bronchial epithelium samples were embedded into paraffin, and 5 μm horizontal sections were cut in a paraffin slicing machine. Samples were dewaxed, washed with water, and then repaired antigen and treated in 3% H₂O₂ for 25 minutes to deactivate endogenous peroxidase. The samples were blocked with 5% fat-free milk for 1 hour and then incubated with primary antibodies (anti-MIP-T3 rabbit polyantibody, diluted 1:200; OriGene, MA, USA) overnight at 4°C in a humidified chamber. They were subsequently incubated with a solution of the goat anti-rabbit IgG HRP-conjugated antibody (diluted 1:200; CWBIO, Guangzhou, China) for 1 hour at room temperature. After chromogenic reaction with DAB staining, the samples were dehydrated, cleared in xylene, and covered with neutral balsam. Image-Pro plus 6.0 software was used to measure mean IOD of the MIP-T3 expression in bronchial epithelium.

Total protein of bronchial epithelium samples was extracted by RIPA buffer (Beyotime, Nantong, China) according to the manufacturer's protocols. After determining concentration by the BCA protein assay kit (Beyotime, Nantong, China), the protein was denatured and stored at −20°C. Samples containing 60 μg protein were subjected to 8% SDS-PAGE electrophoresis and transferred to a PVDF membrane (Weijia, Guangzhou, China). The membrane was blocked with 5% fat-free milk for 1 hour and then blotted with primary antibodies (anti-MIP-T3 rabbit polyantibody, diluted 1:500; OriGene, MA, USA, and anti-GAPDH rabbit polyantibody, diluted 1:2000; CWBIO, Guangzhou, China) overnight at 4°C. They were subsequently incubated with a solution of the goat anti-rabbit IgG HRP-conjugated antibody (diluted 1:5000; CWBIO, Guangzhou, China) for 1 hour at room temperature. Finally, the membranes were processed using the ECL chemiluminescence reaction (Beyotime, Nantong, China) and followed on the RM2016 imaging system. The band density was measured by Image J, and the relative expression of the target protein was compared with GAPDH.