Unfixed slides with the transversally cross-sectioned heart were incubated for 1h with blocking buffer (PBS with 2% BSA and 0.1% Triton X-100) prior to overnight immunostaining at 4°C with rat anti-laminin α2 (4H8–2; Santa Cruz, cat. no. sc-59854) and AlexaFluor635-conjugated phalloidin (Invitrogen, cat. no. A22284). The slides were washed and incubated with anti-rat AlexaFluor488-conjugated IgG (Invitrogen, cat. no. A48262). DAPI (Sigma Aldrich, cat. no. 10-236-276-001) was used to identify nuclei.
For the analysis of cardiomyocyte size, the left ventricle of the heart was identified and imaged on the Nikon C2 confocal microscope with a 20X objective. Phalloidin was used to classify the individual cardiomyocytes (red), while the inverse threshold of laminin α2 was used to determine cardiomyocyte boundaries. Cardiomyocyte size was determined with the Nikon Elements software by using the intersection of inverse laminin and the phalloidin staining. Cardiomyocytes with an area of <100 and >1000 μm2 were excluded. The Feret’s minimal diameter was used to measure cardiomyocyte size and this analysis was performed by using the Nikon Elements software and the “Object Count” function.
For the analysis of cardiomyocyte size, the left ventricle of the heart was identified and imaged on the Nikon C2 confocal microscope with a 20X objective. Phalloidin was used to classify the individual cardiomyocytes (red), while the inverse threshold of laminin α2 was used to determine cardiomyocyte boundaries. Cardiomyocyte size was determined with the Nikon Elements software by using the intersection of inverse laminin and the phalloidin staining. Cardiomyocytes with an area of <100 and >1000 μm2 were excluded. The Feret’s minimal diameter was used to measure cardiomyocyte size and this analysis was performed by using the Nikon Elements software and the “Object Count” function.