Axioscope 40

Hearts dissected from 9 months old wild type (bh^+/+) and mutant (bh^−/−) were fixed in 4% paraformaldehyde, embedded in paraffin, and cross sections were obtained. The sections were stained with hematoxylin and eosin (H&E) (Fisher Scientific) using a standard protocol. Heart tissue sections were observed under an inverted light microscope (Zeiss Axioscope 40) at a magnification of ×5, and details were documented using ×40 magnification. The cytoskeletal structure of heart tissue was observed using Invitrogen™ Texas Red™-X Phalloidin (Thermofisher Scientific), a stain that specifically stains F-actin filaments. Hearts were dissected from nine-month-old adult wild type and bh^−/− mutant zebrafish as described previously (Singh et al., 2016 (link)) and fixed in 4% paraformaldehyde for overnight at 4°C. The heart tissue was processed as per the following protocol: 1X PBS wash for 1 min × 3 times; 0.1% TBST for 5 min × 3 times; 1X PBS wash for 5 min × 2 times. The tissues were incubated with PBS with 1% BSA for 5 min -pre-staining. A total of 5 µL of 6.6 µM of phalloidin conjugated with texas red in methanol was added to 200 µL of detection solution. Tissues were incubated in the dark for 20 min and were washed with 1X PBS × 3 times for 5 min each. Stained tissues were observed using an inverted light microscope (Zeiss Axioscope 40).

Liver specimens were collected from the tissue and kept in 10% formalin solution. After fixation the tissues were processed by serial dehydration, clearing in xylene and then embedded in paraffin wax. 5 µm slices were sectioned and stained by routine Hematoxylin and eosin (H&E) and Masson’s trichrome (M&T). Picro-sirius red was used to stain the liver tissue sections to visualize the collagen content under fluorescence at 475–500 nm^{61 (link)–63 (link)}. Photomicrographs were taken on Zeiss Axioscope A1 with Jenoptik Prog Res C5 camera model and Zeiss Axioscope 40.

To inhibit pigment formation, embryos were treated with phenyl thiourea (PTU) (0.003%). Embryos were anesthetized using 4 mg/mL Tricaine methanesulfonate (Sigma, A5040) and mounted in 0.8%–1% low melting agarose gel (Biorad, 161-3111) in embryo water for imaging. GFP expression was observed, and fish were imaged using an upright Zeiss Axioscope 40 fluorescent microscope (Carl Zeiss, Germany). Image processing was done using Zeiss AxioVision 4.6 and Adobe Photoshop CS software.