Axioimager z3 fluorescent microscope

LV tissue specimens were fixated in 4%-PFA and embedded in paraffin, cut from the apex through the base and stained with Picrosirius red. Slides were scanned to acquire whole slide imaging at × 20 magnification. Total fibrosis area was reported to the total area for each slice.
For the detection of capillary vessels, Isolectin B4 FITC Conjugate (Sigma-Aldrich) staining was performed on paraffin-embedded LV sections of the middle part of the LV. Nuclei were stained with DAPI (Sigma-Aldrich). Capillary density was calculated in a blind fashion by two experimenters as the average number of IB4 + vessels per mm^{2 (link)}. For counting, n = 3 areas were randomly taken both in the border zone of the infarcted remote areas and in the remote zone of one section per left ventricle. Images obtained with a Zeiss Axioimager Z3 fluorescent microscope were analyzed using ImageJ.

At the end of ex vivo experiments, LV were fixated in 4%-PFA and embedded in paraffin. Each LV was cut from apex to base (sections of 4 μM each 150 μM). The paraffin-embedded sections were deparaffinized then rehydrated through an alcohol gradient. Left ventricle sections were incubated with a primary anti α-actinin antibody (1:100, mouse monoclonal; Sigma-Aldrich). Cell nuclei were stained with Hoechst (Life technologies SAS) and endothelial cells with Isolectin B4 (FITC Conjugate; Sigma-Aldrich). After incubation with primary antibodies, sections were washed in PBS, and then incubated (3 h) with secondary antibodies (1:2,000, Jackson ImmunoRes Laboratories, Inc.). Primary and secondary antibodies were diluted in PBS containing 3% BSA and 0.1% Triton X100. Stained sections were mounted in Mowiol (Biovalley). Images were obtained with a Zeiss Axioimager Z3 fluorescent microscope after observation of six different sections of the LV harvested on n = 2 hearts treated by MSC labeled with CM-DiI and analyzed using ImageJ and Adobe Photoshop to prepare the final figures.