Normal sera and igg free bsa

We used a previously well-characterized affinity isolated polyclonal primary antibody raised in rabbit against amino acids 156–205 of the PDE4D protein (SAB4502128; Millipore Sigma Aldrich, Burlington, MA, United States) that recognizes human and rodent PDE4D based on sequence homology. The antibody is highly specific and detects endogenous levels of total PDE4D protein at a band migrating at ∼91 kDa. The antibody is suited for a range of applications, including immunohistochemistry, immunoblotting and ELISA as per manufacturer’s recommendations. The specificity and selectivity of the PDE4D antibody has been previously characterized using immunohistochemistry in myocytes to identify a role of PDE4D-PRKAR1α in cardiac contractility (Bedada et al., 2016 ). The primary antibody was used at 1:200 dilution and was complexed with rabbit-specific goat secondary antibodies. Normal sera and IgG-free BSA were purchased from Jackson ImmunoResearch (West Grove, PA, United States). All chemicals and supplies for electron microscopy were purchased from Sigma Aldrich (St. Louis, MO, United States) and Electron Microscopy Sciences (Hatfield, PA, United States), respectively.

An extensively characterized mouse monoclonal C1q (JL-1) antibody reactive against the collagen-like region (CLR) obtained commercially (ab71940; Abcam, Cambridge, MA, USA) was used for immunoEM studies at 1:200 dilution. The antibody was generated by immunization of C1q^−/− C57BL/6 mice with purified mouse C1q. The antibody has been previously shown to recognize C1q with a high degree of specificity in a myriad of different tissues and cell types by immunoblotting, immunohistochemistry, and immunofluorescence procedures [27 (link)–30 (link)]. The C1q antibody detects a band migrating at ~ 26 kDa in immunoblots. Normal sera and IgG-free BSA were purchased from Jackson Immunoresearch (West Grove, PA, USA). All chemicals and supplies for electron microscopy were purchased from Sigma Aldrich (St. Louis, MO, USA) and Electron Microscopy Sciences (Hatfield, PA, USA), respectively.