Ix81 confocal microscope

Immunofluorescence was mainly performed as described^{56 (link)}. Briefly, freshly dissected bones were fixed in 4% paraformaldehyde for 48 h and incubated in 15% DEPC-EDTA (pH 7.8) for decalcification. Then, specimens were embedded in paraffin or OCT and sectioned at 8 μm. Sections were blocked in PBS with 10% horse serum for 1 h and then stained overnight with mouse-anti-Osteocalcin (Santa Cruz, 1:100, sc-376726), rabbit-anti-Ddah1 (SAB, 1:200, #37368), mouse-anti-Ddah1 (Santa Cruz, 1:100, sc-271337), rabbit-anti-Ddah2 (SAB, 1:200, #38934), mouse-anti-TAZ (Abcam, 1:200, ab242313), rabbit-anti-YAP (Abcam, 1:200, ab52771), and eNOS (Santa Curz, 1:200, sc-376751). Goat-anti-mouse FITC (1:1000; Jackson ImmunoResearch, 705-165-147) and donkey-anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes, A21206) were used as secondary antibodies. DAPI (Cell Signaling Technology, #4083) and DyLight™ 594 Phalloidin (Cell Signaling Technology, #12877) were used for counterstaining. All immunofluorescence experiments were confirmed by at least one independent repeat. An Olympus IX81 confocal microscope or Zeiss LSM-880 confocal microscope was used to image samples.

Immunofluorescence was performed on cryosections of diastole-fixed hearts or on soleus or tibialis anterior muscle and on embryonic cardiomyocytes using standard protocols and the following antibodies: sarcomeric α-actinin (SαA; Sigma), Thymosin β4 (Immundiagnostik), myomesin and cardiac myosin binding protein C (cMyBPC a kind gift of Elisabeth Ehler/Mathias Gautel), cTnT and cTNI, (Abcam); N2B, PEVK, N2A, Tmod1 and Nebulin (kind gifts of Siegfried Labeit). Images were acquired using an Olympus IX81 confocal microscope, a Zeiss AxioImager with ApoTome or a Leica DM6000 fluorescence microscope with Structured Illumination. Adult heart sections were stained with hematoxylin and eosin, using a standard protocol, or Alexa 594-conjugated wheatgerm agglutinin (Invitrogen), according to the manufacturer's instructions. Nuclear counts, cell area and cell counts were performed on sections which had been anonymised and blinded to genotype, using ImageJ software particle analysis.