Ab151552

A pcDNA3.1-Hsp22 plasmid (pHsp22) (Shanghai Genechem Co., Ltd., Shanghai, China) was constructed and used for mouse treatment. LPS (catalog no. L2630) derived from Escherichia coli 0111:B4 was purchased from Sigma-Aldrich Co (USA). ELISA kits for the measurement of IL-1β (MM-0040 M2), IL-6 (MM-1011M1) and TNF-α (MM-0132M1) were obtained from MEIMIAN, Jiangsu Biological Industrial Co., Ltd., China. Primary antibodies used in this study were anti-Hsp22 (Abcam, ab151552), anti-TNF-α (Abcam, ab183218), anti-NLRP3 (CST, #1510), anti-Bax (Abcam, ab32503), and anti-Bcl2 (Abcam, ab32124). The superoxide dismutase (SOD) assay kit (Cat#A001-3-2) and malondialdehyde (MDA) assay kit (Cat#A003-1-2) were procured from the Nanjing Jiancheng Bioengineering Institute, Nanjing, China.

Immunohistochemical staining of the aortas was performed to detect the expression of HSP22, PPAR-γ and intracellular adhesion molecule 1 (ICAM-1) as previously described.²¹ (link) Briefly, the aortas were embedded in paraffin, and 5-μm sections were cut from the embedded blocks. After heat-induced antigen retrieval, the slides were incubated at 4°C overnight with the following primary antibodies: HSP22 antibody (Abcam, ab151552, rabbit monoclonal, at 1:50, USA), PPAR-γ antibody (CST, 2435, rabbit polyclonal, at 1:200, USA) or ICAM-1 antibody (Abcam, ab119871, rat monoclonal, at 1:200, USA). The slides were then incubated with biotinylated IgG (1:250) for 1 h followed by incubation with streptavidin-HRP for 30minat room temperature. DAB was added to each slide for 5 min. The slides were observed under a microscope (Olympus, Japan), and images were analyzed using Image-Pro Plus 6.0 System (Media Cybernetics, USA).⁴¹ (link)