mRNA or protein detection assays and AFOG staining

In situ hybridization and AFOG staining were performed on paraffin sections³⁴. Adult zebrafish hearts were fixed in 4% paraformaldehyde at room temperature for 2 h, dehydrated and then embedded in paraffin and sectioned at 5 μm. Zebrafish brg1 cDNA was cloned from an embryonic cDNA library, of which primer sequences are shown as brg1-F and brg1-R in Supplementary Table 1. Digoxigenin-labelled brg1 probes were synthesized using T7 RNA polymerase (Roche).

For immunofluorescence staining, adult zebrafish hearts were fixed in 4% paraformaldehyde at room temperature for 2 h, dehydrated and then embedded in paraffin and sectioned at 5 μm. The sections were dewaxed in xylene, rehydrated with a series of ethanol and then washed in PBS. To repair the antigen, the citric acid buffer (CW0128S; CWBIO) and the microwave treatment were used. After washing in water and PBS, the sections were blocked in 10% FBS in PBT (1% tween 20 in PBS), and then incubated with primary antibodies (1:50 diluted in PBT containing 10% FBS) overnight at 4 °C. The primary antibodies used for immunofluorescence were anti-BrdU (B8434; Sigma), anti-Mef2c (sc-313; Santa Cruz), anti-GFP (A-11122; Invitrogen), anti-PCNA (18-0110; Invitrogen), anti-GFP (BE2001; EASYBIO), anti-RFP (BE2023; EASYBIO), anti-myosin heavy-chain monoclonal antibody (hybridoma product MF20; Developmental Studies Hybridoma Bank, Iowa City, IA) and The Brg1 JI antibody, which was raised against a glutathione S-transferase–BRG1 fusion protein (human BRG1 amino acids 1,086–1,307)^30,67. The primary antibodies were then washed and sections were incubated with secondary antibodies for 2 h at room temperature. Secondary antibodies (1:100 diluted in PBT) were Alexa Fluor 488 goat anti-mouse IgG (A21121; Invitrogen), Alexa Fluor 488 goat anti-rabbit IgG (A11034; Invitrogen), Alexa Fluor 555 goat anti-mouse IgG (A21424; Invitrogen) and Alexa Fluor 555 goat anti-rabbit IgG (A21428; Invitrogen).

RNAscope (Advanced Cell Diagnostics, Hayward, CA) was performed on 10 μm sections from freshly frozen hearts embedded in O.C.T. Compound (Embedding Medium for Frozen Tissue Specimens to ensure Optimal Cutting Temperature; SAKURA; 4583). Tissues were fixed in pre-chilled 10% neutral buffered formalin, followed by dehydration, then treated with Pretreat 1 for 10 min at room temperature. After Pretreat 1, slides were washed with water and incubated for 30 min at room temperature with Pretreat 4. Following Preteat 4, the RNAscope 2.0 HD Detection Kit Brown was applied for visualizing hybridization signals. Three injured and mock hearts were used for each RNAscope experiment. Immunostaining was performed with primary antibodies (1:50 diluted in PBT containing 10% FBS) incubated overnight at 4 °C.

RNA in situ hybridization, RNAscope in situ hybridization and AFOG staining were analysed and documented under a fluorescence microscope (DM5000B; Leica, Germany). Immunofluorescence images were captured on a confocal microscope (LSM510; Carl Zeiss, Germany). A Zeiss 700 confocal microscope was used for RNAscope with Immunostaining images. The BrdU⁺/Mef2C⁺, PCNA⁺/Mef2C⁺, Brg1⁺, MF20⁺/Brg1⁺, Flk1⁺/Brg1⁺ and Coronin1a⁺/Brg1⁺ were counted manually. Fluorescence intensity was quantitated using MBF Image J.