Mouse anti lc3 monoclonal antibody

Myocardial tissue samples from the remote non-infarcted region were fixed in 10% neutral-buffered formalin, embedded with paraffin, and then sectioned. The paraffin sections, with a thickness of 4 μm, were deparaffinated and rehydrated with xylene and graded alcohol series. The tissue sections were washed in tap water and then incubated with 0.3% H 2 O 2 in methanol for 20 min at room temperature. The tissue sections were washed in phosphate-buffered saline (PBS), blocked with 5% goat serum in PBS, incubated with a mouse anti-LC3 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Beclin1 monoclonal antibody (Abcam, Cambridge, MA, USA) or mouse anti-4HNE monoclonal antibody (EDM Millipore, Billerica, MA, USA), and then incubated with biotin-conjugated anti-mouse IgG (Vector Laboratory, Burlingame, CA, USA). The sections were incubated with avidin and biotinylated horseradish peroxidase macromolecular complex (Vector Laboratory), and stained with 3-amino-9-ethylcarbazole (Vector Laboratory) and hematoxylin. For negative control, the primary antibody was omitted. The samples were examined under a light microscope.

LV tissue paraffin sections were deparaffinized and rehydrated with xylene and graded alcohol series. The tissue sections were washed, blocked with 5% normal goat serum, and subsequently incubated with a mouse anti-LC3 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-Beclin1 monoclonal antibody (Abcam, Cambridge, MA, USA). Sections were incubated with secondary antibody goat anti-mouse IgG-conjugated fluorescin (Vector Laboratories; Burlingame, CA). To identify cardiomyocytes, sections were incubated with mouse anti-α-sarcomeric actin monoclonal antibody (Sigma-Aldrich, St. Louis, MO) and then incubated with goat anti-mouse IgG conjugated TRITC (Sigma). The tissue sections were stained with Hoechst 33258 to visualize nuclei. For negative control, the primary anti-LC3 or anti-Beclin1 antibody was omitted in the assay. Slides were examined under a fluorescence microscopy (ZEISS AXIO).