A7811

Cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 before blocking in 10% normal goat serum/1% BSA (Millipore Sigma). Cells were incubated with primary antibodies (α-actinin [1:200; A7811; Sigma-Aldrich], cardiac troponin T [1:100; ab45932; Abcam], KI67 (1:200; ab8330; Abcam], PHH3 [1:200; 9701; Cell Signaling Technology], and Aurora B kinase [1:100; ab2254; Abcam]) overnight, and detection was mediated by incubation with secondary antibodies (Alexa Fluor antibody conjugates; Thermo Fisher Scientific) for 1 h. DAPI was used as a nuclear marker. Mounting was performed using Fluoromount-G mounting medium (Thermo Fisher Scientific).
To determine cell loss via pathways involving DNA fragmentation, TUNEL assays (Roche) were performed according to the manufacturer’s instructions. In vitro proliferation after FSTL1 supplementation was assessed using Click-iT EdU incorporation kit (Life Technologies). Imaging was performed using a confocal microscope (Leica SP8 X) and image analysis using ImageJ.

Frozen heart sections embedded in OCT compound (Tissue-Tek; Sakura, UAE) were cut into 8 μm sections with a cryostat (Leica, Germany), permeabilized, blocked with Blocking-One (Nacalai Tesque, Japan), and labeled with primary antibodies, followed by fluorochrome-conjugated secondary antibodies. Counterstaining for DAPI (nuclei), phalloidin (F-actin), and wheat germ agglutinin (WGA; cell membrane) was also performed. Sections were covered with a fluorescence mounting medium (Dako, USA) and examined using an inverted fluorescence microscope (BZ-X710, Keyence, Japan), or a confocal scanning system mounted on a IX81 inverted microscope (FV-1000, Olympus, Japan) (Hashimoto et al., 2018 (link)). The primary antibodies used were for Ki67 (clone SP6, Abcam, UK), phospho-histone H3 at Ser-10 (06-570, EMD Millipore, USA), PCM-1 (HPA023370, Sigma-Aldrich), sarcomeric α-actinin (A7811, Sigma-Aldrich), and the FLAG tag (F1804, Sigma-Aldrich). When using mouse-derived antibodies, the Mouse on Mouse (M.O.M.) Basic Kit (Vector, CA, USA) was used. Essentially the same staining protocol was applied for CMs from aged mice isolated with a fixation digestion method (see below). In freshly isolated fetal CMs, fixation was done with 4% paraformaldehyde before permeabilization.

NRVMs cultured on glass coverslips were fixed in paraformaldehyde for 15 min and then washed for three times with 1XPBS. After incubation with blocking solution (5% goat serum and 0.3% Triton X‐100 in 1X PBS) for 1 h, NRVMs were stained with anti‐actinin (1:500, A7811, Sigma‐Aldrich) overnight. Subsequently, NRVMs were incubated with secondary antibodies (1:1,000, A11005 or A11031, Thermo Fisher Scientific). Coverslips were finally mounted with ProLong^® Gold Antifade Reagent with DAPI (P36931, Thermo Fisher Scientific). Images were captured using fluorescence microscope. Surface areas of NRVMs were quantified by ImageJ software. Cytosolic localization of HDAC4 and HDAC5 was calculated and presented as percentage (Toth et al, 2018).

Cultured cells were fixed with 4 % paraformaldehyde for 15 min, washed twice with PBS, and subsequently permeabilized with 0.5 % Triton X-100 (X100; Sigma-Aldrich) for 10 min. The washed cells were blocked for 30 min at room temperature with 1 % BSA (A9418; Sigma-Aldrich), and then incubated overnight at 4 °C with primary monoclonal mouse anti-alpha-actinin (A7811; Sigma-Aldrich) antibody diluted in 0.2 % BSA solution. After three serial washes with PBS, cells were incubated with AlexaFluor 594 conjugated goat anti-mouse antibody for 1 h at room temperature, and then washed and incubated in 300 nM DAPI for 5 min for nuclear counterstain.

For immunohistochemistry staining, heart cryosections were fixed with 4% paraformaldehyde, permeabilized and blocked with Protein Block Solution (DAKO, Carpinteria, CA) containing 0.1% saponin (Sigma, St Louis, MO), and then incubated with the following antibodies overnight at 4 °C: mouse anti-alpha sarcomeric actin (1:100, a7811, Sigma), rabbit anti-CD45 (1:100, ab10559, Abcam, Cambridge, United Kingdom), mouse anti-Actin, α-Smooth Muscle antibody (1:100, A5228, Sigma), rabbit anti-Ki67 (1:100, ab15580, Abcam), rabbit anti-CD3 (1:100, ab16669, Abcam) and mouse anti-CD8 alpha (1:100, mca48r, abd Serotec, Raleigh, NC ). FITC- or Texas-Red secondary antibodies (1:100) were obtained from Abcam Company and used for the conjunction with these primary antibodies. For assessment of cell apoptosis, heart cryosections were incubated with TUNEL solution (Roche Diagnostics GmbH, Mannheim, Germany) and counter-stained with DAPI (Life Technology, NY, USA). For assessment of angiogram, heart cryosections were incubated with Lectin (FL-1171, Vector laboratories, Burlingame, CA, USA). Images were taken by an Olympus epi-fluorescence microscopy system.