Horseradish peroxidase hrp conjugated rabbit anti mouse igg

ASMCs were identified by α-smooth muscle actin immunocytochemical staining, as previously described (18 (link),19 (link)). Briefly, ASMCs were seeded onto sterile glass coverslips and grown to 70% confluence. Cells were fixed in 4% paraformaldehyde for 20 min, and the sections were reacted with 3% H₂O₂ (Guangzhou Whiga Technology Co., Ltd.) After a rinse in phosphate-buffered saline (PBS), the sections were blocked with 2% bovine serum albumin (BSA; Guangzhou Whiga Technology Co., Ltd.) and incubated with monoclonal mouse anti-α-smooth muscle actin antibody (1:300; Thermo Fisher Scientific, Inc.) at 4°C overnight for immunolabeling. Subsequently, the sections were incubated with a secondary horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:300; Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 37°C and then washed three times with PBS. The peroxidase activity was visualized by a color reaction using 3,3′-diaminobenzidine (1 ml; Wuhan Boster Biological Technology, Ltd., Wuhan, China) as the substrate. The slides were then counterstained with hematoxylin (Wuhan Boster Biological Technology, Ltd.), mounted and examined under a microscope (Olympus Corp., Tokyo, Japan) using AxioVision 4.1 software (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA).

Protein was isolated from cells and extracted using radioimmunoprecipitation assay lysis buffer (Beijing Leagene Biotech Co., Ltd.), and the total protein was quantified using a bicinchoninic acid detection assay kit (Shanghai Yeasen Biotechnology, Co., Ltd.). Proteins (20 _µg/lane) were separated via 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in 5% dried skimmed milk in TBS buffer at 37°C for 1 h and incubated overnight at 4°C with antibodies against TLR3 (1:700; ab62566, Abcam), TLR4 (1:600; ab13556, Abcam), TLR5 (1:800; ab62460, Abcam), p65 (1:1,000; ab32536, Abcam), phosphorylated (p)-p65 (1:600; ab86299, Abcam) and β-actin (1:1,000; MAB8969, R&D Systems, Inc.). Then, the membranes were washed in TBS-0.1% Tween-20 three times, and incubated at room temperature for 1.5 h with the following secondary antibodies: Mouse anti-rabbit IgG, (1:6,000; cat. no. 3678; Cell Signaling Technology, Inc.); horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:7,000; cat. no. 58802; Cell Signaling Technology, Inc.) and HRP-conjugated rabbit anti-goat IgG (1:6,000; sc-2768; Santa Cruz Biotechnology, Inc.). Bands were visualized using enhanced chemiluminescence detection reagent (Amersham; GE Healthcare) and imaged using an iBright™ CL1000 imaging system (iBright analysis software, version 1.2.0; Thermo Fisher Scientific, Inc.).