Blood was collected in heparin-containing vials when mice were killed, centrifuged at 4°C (3,000 rpm in 20 min), and stored at −80°C. Plasma Gal-3 levels were detected by a mice Galectin-3 Quantikine ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA) in twin duplicates wells, following protocols provided by the manufacturer. The detection range of the plasma Gal-3 was 8.19–2,000 pg/mL, and the limit of detection was 6 pg/mL.
Paraffin-embedded LV sections (6 μm) were prepared and used for Gal-3 immunofluorescent staining within mice. For Gal-3 immunofluorescent staining, after dewaxed, heat-induced antigen retrieval and permeabilization were carried out (with 10 mM of Na-citrate buffer containing 0.05% Tween 20; pH 6.0; 95°C for 25 min) followed by blocking with DAKO Protein Block (X0909, Agilent, 1 h at room temperature). Sections were incubated with primary goat anti-mouse Gal-3 (1:100, AF1197, R&D Systems) overnight at 4°C, then they were incubated with the secondary antibody, Alexa Fluor 594 donkey anti-goat IgG (1:200, A11058, Invitrogen by Thermo Fisher Scientific). The cardiomyocyte boundary was revealed by wheat-germ-agglutinin FITC staining (1:80, FL-1021, Vector Labs, 1 h at room temperature). Images were acquired with an Olympus BX61 fluorescent microscope.
Paraffin-embedded LV sections (6 μm) were prepared and used for Gal-3 immunofluorescent staining within mice. For Gal-3 immunofluorescent staining, after dewaxed, heat-induced antigen retrieval and permeabilization were carried out (with 10 mM of Na-citrate buffer containing 0.05% Tween 20; pH 6.0; 95°C for 25 min) followed by blocking with DAKO Protein Block (X0909, Agilent, 1 h at room temperature). Sections were incubated with primary goat anti-mouse Gal-3 (1:100, AF1197, R&D Systems) overnight at 4°C, then they were incubated with the secondary antibody, Alexa Fluor 594 donkey anti-goat IgG (1:200, A11058, Invitrogen by Thermo Fisher Scientific). The cardiomyocyte boundary was revealed by wheat-germ-agglutinin FITC staining (1:80, FL-1021, Vector Labs, 1 h at room temperature). Images were acquired with an Olympus BX61 fluorescent microscope.