Immunohistochemistry Analysis of Heart Tissue

An immunohistochemical analysis were performed as described previously [40 (link),41 (link)]. The heart tissues were fixed in 10% buffered formaldehyde and 7 μm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H₂O₂ in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 20 min. Non-specific adsorption was minimized by incubating the section in 2% normal goat serum in phosphate-buffered saline for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin. The sections were probed with the following primary antibodies: anti-CD4 (Santa Cruz Biotechnologies, sc-13573, Dallas, TX, USA) and anti-CD68 (Santa Cruz Biotechnologies, sc-20060). The slides were then washed with PBS and incubated with a secondary antibody. Specific labeling was achieved with an avidin–biotin–peroxidase complex and biotin-conjugated goat anti-rabbit immunoglobulin G (Vector Lab, Milan, Italy) [42 (link)]. The stained sections were observed using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy).