Anti α sarcomeric actin

Immunohistochemistry and histological staining were performed on 10 μm heart sections as previously described (27 (link)). The antibodies used in this manuscript are listed below:
anti-Trpc6 (OST00081W, Osenses)
anti-Trpm (T2780, Sigma)
anti-Mef2c (ab197070 Abcam)
anti-α-Sarcomeric actin (A2172, Sigma)
EdU labelling was performed according to the manufacturer's instructions (Click-iT EdU Kit C10337, Molecular Probes). Acid Fuchsin-Orange G (AFOG) staining was performed as previously described (28 (link)) and the size of the scar area was calculated using ImageJ software. T-test statistical analyses was performed using GraphPad Prism. Alkaline phosphate staining was performed on whole-mount heart as previously described (27 (link)).

Heart tissue samples were treated as previously described [10 (link)]. The expression of mannose-6-phosphate receptors was visualized by using IGF-II Receptor/CI-M6PR (D8Z3J) polyclonal antibody (1:1000, Cell Signaling Techology, Danvers, MA, USA). Anti-α-sarcomeric actin (1:500, Sigma-Aldrich) antibody was used for normalization. Signal was visualized by using a secondary horseradish peroxidase-labeled goat anti-mouse antibody (goat anti-mouse IgG-HRP 1:5000, SantaCruz Biotechnology, Dallas, TX, USA) and enhanced chemiluminescence (ECL Clarity Bio-Rad, Hercules, CA, USA). The purity, as well as equal loading (40γ) of the protein, was determined by Nanodrop One (Thermofisher, Waltham, MA, USA). To normalize target protein expression, the band intensity of each sample was determined by densitometry with Image J software. Next, the intensity of the target protein was divided by the intensity of the loading control protein. This calculation adjusts the expression of the protein of interest to a common scale and reduces the impact of sample-to-sample variation. Relative target protein expression can then be compared across all lanes to assess changes in target protein expression across samples. Digital images of the resulting bands were quantified by the Image Lab software package (Bio-Rad Laboratories, Munchen, Germany) and expressed as arbitrary densitometric units.