Preheated bouin s solution

Zebrafish hearts were extracted and fixed in 4% (wt/vol) paraformaldehyde (Alfa Aesar, MA) at room temperature for 1 hr. Collected hearts were subsequently cryopreserved with 30% (wt/vol) sucrose, followed by immersed in OCT (Tissue-Tek, Sakura Finetek, Torrance, CA) and stored at −80°C immediately. 10 μm cryosections were collected for histological analysis. AFOG staining was applied for the visualization of healthy CMs in orange, collagens in blue and fibrins in red. In brief, samples were incubated in preheated Bouin’s solution (Sigma) at 58°C for 2 hr post fixation and subsequently immersed in 1% phosphomolybdic acid (Sigma) and 0.5% phosphotungstic acid solution (Sigma) at room temperature for 5 min for mordanting. Samples were then incubated with AFOG solution containing Aniline Blue (Sigma), Orange G (Sigma), and Acid Fuchsin (Sigma) for color development. Quantification was done by measuring the scar area in each heart from five discontinuous sections including the one with the largest scar as well as the two sections at the front and the two sections at the back as previously described (Lai et al., 2017 (link)). Statistic was performed on Prism 9 using Student’s t-test.

For aniline blue staining, we modified the conventional trichrome staining protocol. Briefly, slides were deparaffinized and hydrated with deionized H₂O followed by incubation in pre-heated Bouin's solution (Sigma) for 15 minutes. Next, slides were cooled at room temperature and washed under running tap water. Tissue sections were then incubated in working phosphotungstic-phosphomolybdic acid solution (Sigma) for five minutes followed by aniline blue (Sigma) for 10 minutes and rinsed in 1% acetic acid (SRL) for 2 minutes. Finally, slides were thoroughly washed with deionized H₂O and dehydrated through sequential alcohol grading. They were then cleared in xylene and mounted with permanent mounting media (Vector Lab). Stained slides were observed under a Leica DM500 light microscope, and images were taken at 10× and 40× magnifications. The percentage of collagen stained area (aniline blue) was quantified using the automated ImageJ program in conjunction with the threshold plug-in [43 (link)].