Vectashield conjugated 4 6 diamidino 2 phenylindole dapi

Human myocardial LV samples were fixed in 4% formalin, embedded in paraffin, cut into 5-µm sections, and mounted on superfrost glass slides. Sections were kept at 60°C overnight, deparaffinized with xylol followed by washing in 100%, 96%, 80%, and 70% ethanol. The samples were then blocked with phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 15 min at RT. After blocking, the sections were incubated for 120 min at RT with the primary antibodies (described in the Western Blot Analysis section above) in the same buffer solution, and then with Alexa-conjugated secondary antibody (Invitrogen, USA) for 60 min at RT [21] (link). Finally, the sections were rinsed in PBS, mounted in Vectashield-conjugated 4′,6-diamidino-2-phenylindole (DAPI) for identifying the nucleous (Vector Laboratories, Burlingame, CA, USA), and examined under an Olympus BX50 fluorescence microscope (Tokyo, Japan). The images were processed by ImageJ (v. 1.4.3.67) software.

Human myocardial LV samples were fixed in 4% formalin, embedded in paraffin, cut into 5-μm sections, and mounted on SuperFrost glass slides. Sections were maintained at 60°C overnight, deparaffinized using xylol, and then washed in 100%, 96%, 80% and 70% ethanol. Next, the samples were blocked with PBS containing 1% bovine serum albumin (BSA) for 15 min. at room temperature (RT). After blocking, the sections were incubated for 120 min. at RT with primary antibodies (described in the ‘Western blot analysis’ section above) diluted in the blocking buffer and were then incubated with Alexa-conjugated secondary antibodies (Invitrogen, New York, USA) for 60 min. at RT 17 (link). Finally, the sections were rinsed in PBS, mounted in Vectashield-conjugated 4′,6-diamidino-2-phenylindole (DAPI) to identify nuclei (Vector Laboratories, Burlingame, CA, USA), and examined under an Olympus BX50 fluorescence microscope (Tokyo, Japan). The images were processed using the ImageJ software (v. 1.46r; National Institutes of Health, Bethesda, MD, USA).