Immunohistostaining of Aortic Lamins

Immunohistostaining was performed following the protocol previously described^{7 (link)} with modifications, and by using mouse monoclonal anti-lamin A/C (MABT538, clone 2E8.2, Millipore Sigma; 1:75 dilution) antibody or rabbit polyclonal anti-progerin antibody (Collins, custom; 1:75 dilution). Briefly, ascending aorta sections were dewaxed and rehydrated, and the antigens were retrieved by heating in EDTA buffer (1 mM, pH 8.0) for 2 min in a pressure cooker. Tissue sections were blocked in TBS buffer containing 10% donkey serum and 1% BSA, and then incubated with a Mouse-on-Mouse blocking reagent (Vector Laboratories) to reduce endogenous mouse antibody binding. Slides were incubated with the above primary antibody overnight at 4 °C. After washing thoroughly in TBS, the sections were then incubated with donkey anti-mouse Alexa Fluor 594-conjugated or donkey anti-rabbit Alexa Fluor 488-conjugated secondary antibodies (ThermoFisher Scientific; 1:3000 dilutions). All tissue sections were mounted in DAPI-containing medium (Vector Laboratories). Fluorescence images were captured by a confocal microscope system (Zeiss LMS 880) with 40x water lens.