Formalin-fixed, paraffin-embedded hearts from sham and HF mice were sectioned at 5 μm thickness, deparaffinized, and rehydrated. Histological staining was performed as previously described.6 (link), 21 (link), 23 (link) Masson’s trichrome was used to evaluate tissue fibrosis and Alexa Fluor 488–conjugated wheat germ agglutinin (Invitrogen) to assess myocyte area, as quantified from 5 to 6 high-power fields per section in non-infarcted myocardium using Metamorph software version 6.3r5 (Molecular Devices).
To evaluate for tissue abundance of CD4+ and CD8+ T-cells, heart sections were embedded in OCT compound (Tissue-Tek OCT), and then kept at −80°C until sectioning. Sections (7 μm thickness) were fixed with 4% paraformaldehyde in PBS, and labeled with either rat anti-mouse CD4 (Clone GK1.5; eBiosciences) or CD8 antibody (Clone 4SM15; eBiosciences). Goat anti-rat antibody conjugated with Alex Fluor 555 and 488 (Life Technologies) were used as secondary antibodies for CD4 and CD8, respectively, and fluoroshield-containing DAPI (Sigma-Aldrich) was used as the mounting medium. CD4+ or CD8+ cells were quantified using confocal microscopy from 5–6 sections and 3–4 mice from each group. All measurements were conducted in a blinded manner. Confocal microscopy was performed on an LSM710 microscope (Zeiss).
To evaluate for tissue abundance of CD4+ and CD8+ T-cells, heart sections were embedded in OCT compound (Tissue-Tek OCT), and then kept at −80°C until sectioning. Sections (7 μm thickness) were fixed with 4% paraformaldehyde in PBS, and labeled with either rat anti-mouse CD4 (Clone GK1.5; eBiosciences) or CD8 antibody (Clone 4SM15; eBiosciences). Goat anti-rat antibody conjugated with Alex Fluor 555 and 488 (Life Technologies) were used as secondary antibodies for CD4 and CD8, respectively, and fluoroshield-containing DAPI (Sigma-Aldrich) was used as the mounting medium. CD4+ or CD8+ cells were quantified using confocal microscopy from 5–6 sections and 3–4 mice from each group. All measurements were conducted in a blinded manner. Confocal microscopy was performed on an LSM710 microscope (Zeiss).