Mouse on mouse m o m basic kit

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.

For tissue analyses, frozen heart sections embedded in OCT compound (Tissue-Tek^®; Sakura, UAE) were cut at 8 µm sections with a cryostat (Leica, Germany), permeabilized, blocked with Blocking-One (Nacalai Tesque, Japan), and double-labeled with primary antibodies for sarcomeric α-actinin (Sigma-Aldrich) and Fam64a. The latter antibody was raised against a synthetic peptide corresponding to residues 102–114 of mouse Fam64a (CQSGTKWLMETQV). Samples were then labeled with fluorochrome-conjugated secondary antibodies (Thermo-Fisher) as described^{25 (link), 26 (link)}. When necessary, DAPI staining for DNA or phalloidin staining for F-actin filaments was also performed. The same protocol was applied for cultured cells, except that fixation with 4% paraformaldehyde was first performed before permeabilization. Primary antibodies used were against Ki67 (Abcam), phospho-histone H3 at Ser-10 (EMD Millipore, MA, USA), sarcomeric α-actinin (Sigma-Aldrich), and Fam64a (Bioss Antibody, GA, USA). Another Fam64a antibody, which was raised as stated above, was also used. When using mouse-derived antibodies, the Mouse on Mouse (M.O.M.^™) Basic Kit (Vector, CA, USA) was used. Cells or sections covered with fluorescence mounting medium (DAKO, CA, USA) were examined using a confocal system mounted on a IX81 inverted microscope (Olympus).