Bouin s solution

All procedures conducted during this trial were in accordance with approval by the Deakin University Animal Ethics Committee (Permit No. B29-2017), and were compliant with the guidelines outlined in the Australian code for the care and use of animals for scientific purposes (2013). This study used 176 (39.72 g ± 1.03) mixed-sex, freshwater acclimated juvenile Atlantic salmon from Salmon Enterprises of Tasmania Pty. Ltd. (SALTAS), Wayatinah, Tasmania. All fish were randomly selected for use in this study from a population that was being maintained under commercial conditions. Sampling was conducted over three consecutive days. Each fish was euthanized by a lethal dose of AQUI-S (AQUI-S, New Zealand). Once euthanized, mass was recorded for each fish followed by collection of dorsal muscle tissue and a single gonad. Muscle tissue was immediately placed in 500 µl of RNAlater (Merck, Germany) and stored at ambient temperature for 5–7 days, then −80 °C for 12 months until time of analysis. The gonad was fixed in Bouin’s solution (Merck, Germany) for 5–7 days, then stored in 70% ethanol until time of processing.

Immunohistochemistry was performed according to standard protocols except the fixation process. Larvae were dissected in ice-cold PBS. Filet preparations were fixed in Bouin's Solution (Sigma-Aldrich) for 3 min at room temperature, while the adult brains were fixed in 2% PFA for 90 min at room temperature. The following antibodies were used: mouse anti-Repo (DSHB, 1:5), rat anti-DN-Cadherin (DSHB, 1:10), mouse anti-V5 (Thermo Fisher Scientific, 1:500), rabbit anti-GFP (Thermo Fisher Scientific, 1:1000), rabbit anti-dsRed (Clontech, 1:1000), goat anti-HRP-Cy5 (Dianova, 1:200). Rabbit anti-Rumpel peptide antibodies were generated against the C-terminal domain of Rumpel (Pineda, Berlin, 1:500). Secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 568 or Alexa Fluor 647 were used (Thermo Fisher Scientific, 1:1000). Ovaries were dissected in ice-cold PBS and fixed in 4% PFA for 15 min. Immunohistochemistry for ovaries was performed as described (Bogdan et al., 2005 (link)). Alexa Fluor 568 Phalloidin (Thermo Fisher Scientific, 1:100) and DAPI (Thermo Fisher Scientific, 1:1000). Confocal microscopy data was generated using a Zeiss 710 or 880 LSM or Leica TCS SP8 DLS. Images were acquired using either the Zeiss LSM ZEN imaging software, or the LSM LAS X software and analyzed using Fiji (Schindelin et al., 2012 (link)).

ESCs and MSCs were fixed with Bouin’s solution (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 4 °C and stored. Fixed cells were blocked, permeabilized and incubated with the primary antibodies overnight in TCT buffer (0.25% Carrageenan and 0.1% Tween-20 in Tris-HCl buffer pH 7.8). The primary antibodies used were: Sox2 (1:100, Abcam, Cambridge, UK), E-Cadherin (1:15, BD Biosciences, San Jose, CA, USA), CD44 (1:100, Abcam), βI Integrin (1:100, Abcam), Musashi (1:200, Abcam) and Vimentin (1:100, Abcam). After rinsing, appropriate secondary antibodies conjugated to Alexa fluorophores 488 or 598 (Molecular Probes, Invitrogen, Carlsbad, CA, USA) were diluted at 1:500 in TCT solution and incubated for 1 hr at room temperature. After rinsing, cells were mounted in Vectashield hardset mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector laboratories) to counterstain nuclei. Images were acquired with a Leica DMI 6000B microscope.

In order to assess fibrosis (% collagen deposition), a Sirius Red/Fast Green staining was prepared. Briefly, paraffin-embedded heart sections were stained with Sirius Red then counterstained with Fast Green. Slides were deparaffinized in a series of degrading alcohol gradient and fixed in Bouin’s solution (Sigma) for 1 h. Then, slides were washed in running water until Bouin’s cleared and then placed in 0.1% Fast Green/ddH₂O for non-collagenous proteins. Slides were then washed in 0.1% acetic acid before staining with 0.1% Sirius red solution for 30 min and then dehydrated and mounted with xylene-based mounting medium. Collagen was semi-quantified using a pixel based method with the use of Adobe Photoshop CC 2017, originally described by Underwood et al. Red pixels were positively selected and summed for a total number of red pixels representing collagen. Nonbackground (green) pixels and red pixels were summed for the total number of heart pixels. Collagen content was measured by calculating the percentage of red pixels by total heart pixels. Consistency was ensured by using the same red colour palette, green colour palette, and by processing all tissues simultaneously.