Cardiac troponin t

Frozen sections and cell climbing slices were fixed in cold acetone, washed with PBS, and then incubated with 3% BSA in PBS for 60 min to block nonspecific binding of the antibodies. Thereafter, the samples were incubated with primary antibodies specific to mouse CD68 (diluted 1:200; Serotec), Ly6G (diluted 1:50; BD Biosciences), CD4 (diluted 1:200; eBioscience), CD31 (diluted 1:500; BD Biosciences), cardiac troponin T (diluted 1: 100; Santa Cruz Biotechnology), or α‐actinin (diluted 1:1,000; Sigma‐Aldrich) overnight at 4°C. Afterward, the slides were washed with PBS three times and incubated with corresponding secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 555 (diluted 1:1000; Invitrogen, Carlsbad, CA, USA) at room temperature for 2 h. Sample was mounted in ProLong Gold antifade reagent with DAPI (Invitrogen). All of the immunofluorescence images were captured and analyzed using a laser‐scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). For the MI model, at least 10 random images from three different sections were obtained from each animal. For cells growing on the glass slide, at least five random fields were taken in the central region of each sample (Shen et al, 2016).