Mouse anti hsp27 monoclonal antibody

Proteins were extracted from the aortic media specimens with a commercial lysis buffer (CelLytic, Sigma-Aldrich Corporation). Western blot was performed with standard procedures. A mouse anti-HSP27 monoclonal antibody (1:1000, Cell Signaling Technology) and a horseradish peroxidase-labeled secondary antibody (1:5000, Santa Cruz Biotechnology) were used. The blots were quantified using the Image J software (National Institutes of Health).

Formalin-fixed, paraffin-embedded aortic sections were deparaffinized and rehydrated before antigen retrieval. The sections were incubated with mouse anti-HSP27 monoclonal antibody (1:50, Cell Signaling) at 4 °C overnight, and then incubated with peroxide-conjugated anti-mouse IgG secondary antibody and stained with 3, 3-diaminobenzidine using the Vectastain ABC kit (Vector Laboratories). For double immunofluorescence staining, the sections were incubated with mouse anti-HSP27 antibody (1:50, Cell Signaling) and rabbit anti-SM22α antibody (1:200, Abcam) at 4 °C overnight. Sections treated only with normal IgG were used as negative controls. Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies (Invitrogen) were used.