Rabbit anti sm22α antibody

Murine and human aortas were fixed in 4% formalin and embedded in paraffin according to standard protocols. Hematoxylin and eosin (H&E) staining was performed for morphological assessment. For immunohistochemistry, a rabbit anti-FoxO3a antibody (1:200; Cell Science, USA), donkey anti-α-SMA antibody (1:200; Abcam, UK), rabbit anti-SM-22α antibody (1:200; Abcam, UK), and rabbit anti-Osteopontin antibody (1:200; Abcam, UK) were used. Paraffin sections of murine and human aortas were dewaxed and hydrated, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min. The sections were incubated with 5% bovine serum for 1 h at room temperature to block nonspecific binding sites followed by primary antibody at 4 °C overnight and secondary antibody (1:500; Cell Science, USA) for 60 min. All specimens were stained with DAB and hematoxylin staining solution. A minimum of three microscopic fields of stained slides were randomly observed by two independent researchers who were unaware of the group information.

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.