FIGURE 4

Collagen distribution and osteoblastic differentiation in young and aged zebrafish valves. (A–D) Images of cleared AVs (A, B) and 3D renders of a single leaflet (C, D) from young (A, C) and aged (B, D) zebrafish, labelled with an anti‐Collagen I antibody. The major leaflet (MaL) shown in C, D is marked in yellow on A, B, respectively. White arrowheads in B, D indicate cystic regions devoid of collagen. (E–H) Images (E, F, higher magnification in F′) and 3D renders (G, H) of the cleared AV labelled with an anti‐Collagen I antibody from young (E, G) and aged (F, H) Tg(sp7:mCherry) transgenic zebrafish. White arrowheads indicate mCherry+ regions outside the valve. (I–L) Images (I, J, higher magnification in J′) and 3D renders (K, L) of the cleared BV labelled with an anti‐Collagen I antibody from young (I, K) and aged (J, J′, L) Tg(sp7:mCherry) transgenic zebrafish. Cyan arrowheads indicate mCherry+ areas within the valve. The boxed regions in F and J indicated the approximate position of F′ and J′. (M) Quantification of the total mCherry+ area within the whole valve of young and aged fish. Due to low sample numbers for individual valves, both the AV and BV are plotted together. Points for aged fish: AV, magenta; BV, blue. Individual leaflets are outlined with white dashed lines in A, B, E, F, I, J, F′, J'. A, atrium; BA, Bulbus arteriosus; MaL, major leaflets; miL, minor leaflets; V, ventricle. Scale bars: A–L = 100 μm; F′, J′ = 50 μm.