Quantifying Necrotic Cardiomyocytes via Evans Blue Assay

Plasma membrane rupture, a major characteristic of necrosis that is not observed during apoptosis in vivo, was evaluated by Evans blue dye uptake assay as described previously (14 (link)). Briefly, mice received a single intraperitoneal injection of Evans blue (10 mg/ml, A16774, Alfa Aesar, 100 μg/g body weight) 16 h prior to I/R. At 24 h post I/R, mice were sacrificed and perfused retrogradely with 10 ml PBS. The heart was harvested and embedded in optimal cutting temperature (OCT) compound (Sakura), snap frozen in liquid nitrogen, and cut into 5-μm cryosections. Heart sections were stained with mouse anti-cardiac troponin T (cTnT, MS-295-P, Thermo Scientific, 1:100) to identify necrotic cardiomyocytes (Evans blue⁺/cTnT⁺). The percentage of necrotic cardiomyocytes was quantified with ImageJ.