For whole-mount immunolabeling, hearts were permeabilized in PBS with 0.1% Tween 20 (PBST), blocked for 1 hr in PBST/10% goat serum, and incubated overnight at 4°C with primary antibody in blocking buffer. Primary antibodies anti-PECAM-1 (Mec13:3, Pharmingen) and anti-SM22 α (Abcam) were diluted in blocking buffer at 1:50 and 1:250, respectively. Hearts were washed several times in PBST (1-hr washes) before incubation overnight at 4°C with secondary antibody (Alexa Fluor 594 conjugate, Life Technologies). Fluorescent images were captured on a Zeiss Axio Lumar.V12 stereomicroscope.
Immunolabeling of 10-μm paraffin sections and 12-μm frozen sections was carried out according to standard protocols. Briefly, sections were incubated in blocking buffer for 1 hr at room temperature (PBS/10% BSA/1% goat serum for wax sections and PBS/10% BSA/10% goat serum/0.1% Triton X-100 for cryosections) followed by primary antibody in blocking buffer at 4°C overnight. Secondaries were Alexa Fluor conjugated antibodies (Life Technologies, Abcam, Jackson ImmunoResearch), and DNA was counterstained using DAPI (Sigma). Images were captured on a Zeiss Axioimager Z1 microscope. Further details, including details of antigen retrieval and antibodies used, can be found inSupplemental Experimental Procedures .
Immunolabeling of 10-μm paraffin sections and 12-μm frozen sections was carried out according to standard protocols. Briefly, sections were incubated in blocking buffer for 1 hr at room temperature (PBS/10% BSA/1% goat serum for wax sections and PBS/10% BSA/10% goat serum/0.1% Triton X-100 for cryosections) followed by primary antibody in blocking buffer at 4°C overnight. Secondaries were Alexa Fluor conjugated antibodies (Life Technologies, Abcam, Jackson ImmunoResearch), and DNA was counterstained using DAPI (Sigma). Images were captured on a Zeiss Axioimager Z1 microscope. Further details, including details of antigen retrieval and antibodies used, can be found in