Free-of-Acrylamide SDS-based Tissue Clearing (FASTClear) for three dimensional visualization of myocardial tissue

Figures

• 

  Figure 1. (A) Schematic representation of FASTClear protocol on freshly prepared or fixed myocardial slices and (B) combination of FASTClear protocol with immunostaining.

• 

  Figure 2. FASTClear provides an efficient myocardial tissue clearing protocol. (A–C) representative images of a myocardial slice before FASTClear, after SDS buffer treatment and after FASTClear protocol. (D) During tissue clearing the architecture and proportions of the myocardium is preserved and no changes in the inter-capillary distance to capillary diameter ratio were observed. Error bar = SEM. P > 0.05 T-test. (E–G) Orthogonal view of a myocardial slice after 48 hours antibody incubation, the labelling is limited to the surface of the preparation. (H–J) Orthogonal view of a myocardial slice after 1 week of antibody incubation. The labelling is homogeneously distributed and reached the core of the preparation. At least 3 slices were used for each experiment.

• 

  Figure 3. SHG imaging of Collagen type 1 in canine and human myocardial slices. Lower magnification of canine (A,C) and human (B,D) myocardial tissue. The autofluorescence (red) is used to show the structure of the myocardium; the collagen fibres (green) are located within the cardiac muscle fibres. The white arrows indicate the collagen in proximity to a large vessel. Higher magnification of healthy dog samples (E,G): the collagen is organised in strands between the muscle fibres; in human heart failure samples (F,H) the regular structure is partially lost and the collagen is less organised. At least 3 slices were used for each experiment.

• 

  Figure 4. Immunostaining and imaging of micro and macrovasculature in cardiac tissue. (A) Two dimensional image of capillaries labelled with IsolectinB4. (B–D) Three dimensional reconstruction of myocardial tissue microvasculature at different magnifications. (E) Two dimensional image of larger vessels labelled with Vimentin. (F–H) Three dimensional reconstruction of myocardial tissue macrovascular network at different magnifications. At least 3 slices were used for each experiment.

• 

  Figure 5. Combination of collagen type 1 imaging and macrovasculature staining. (A) Three dimensional reconstruction of the collagen (green) distribution in proximity to large vessels (vimenitin positive, red). (B,C) 3D and 2D higher magnification shows how the collagen separates myocardial muscle fibres (white arrows) but also surrounds larger vessels (yellow arrows). At least 3 slices were used for each experiment.