Histomount mounting medium

Neural structures were visualised from flattened atrial tissue prepared from pressure distended whole hearts [14 (link)].Whole hearts were illuminated by a fibre optic light guide system (KL1500 LCD, Schott UK) and imaged using a Leica M80 microscope (Leica Microsystems, Germany) at magnifications between ×0.75–6.
The walls of the atria and interatrial septum were separated from the ventricles along the atrioventricular groove and dissected into distinct regions:-1.

The heart hilum

2.

The wall of the conus arteriosus(CA) and left ventricle (LV)

3.

The region ventral to the roots of the pulmonary veins(PVs)

4.

The root of the right cranial vein (RCV)

Wholemount preparations were pinned flat and dehydrated through a series of graded ethanol solutions (70 %, 90 % and 100 %). Sections were immersed in xylene (Fisher, UK) (30 mins to 2 h) and mounted using Histomount mounting medium (National Diagnostics, UK) and covered with a coverslip for microscopic analysis.

The tissue sections were initially deparaffinized in an oven at 65°C for 2 h, immersed in xylene 3 times and subsequently heated to 100°C in 10 mM citrate buffer (Origene Technologies, Inc., Beijing, China), pH 6.0, for 30 min. Sections were blocked with 1% bovine serum albumin (A1933; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and 5% normal goat serum (S-1000; Vector Laboratories, Inc., Burlingame, CA, USA) in PBS plus 0.04% tween 20 at room temperature for 1 h. Sections were subsequently incubated with primary anti-LGR5 (1:100; ab75850; Abcam, Cambridge, UK), anti-GATA6 (1:150; sc-9055; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-catenin antibodies (1:200; 51067-2-AP; ProteinTech Group, Inc., Chicago, IL, USA) at 4°C overnight. Samples were treated with a 2-step plus Poly-HRP anti-mouse/rabbit IgG detection system (cat. no. PV-9000; Origene Technologies, Inc.) and 3,3′-diaminobenzidine tetrahydrochloride (cat. no. ZLI-9032; Origene Technologies, Inc.), according to manufacturer's protocols. Finally, samples were counterstained with Mayer's hematoxylin (cat. no. MHS16; Sigma-Aldrich; Merck KGaA) at room temperature for 5 min and mounted with Histomount mounting medium (HS-103; National Diagnostics, Atlanta, GA, USA).