Alexa 594 donkey anti goat

Sections prepared as described above were thawed, dried at room temperature (RT, 15 min) and fixed with ice-cold 2% paraformaldehyde (w/v, Carl Roth, Karlsruhe, Germany) in PBS (phosphate-buffered saline: 137 mM NaCl, 10 mM Na₂HPO₄, 2.7 mM KCl, 1.8 mM KH₂PO₄) for 6 min. After washing (3x PBS, 5 min, RT), slices were blocked with 5% (w/v) BSA (bovine serum albumin), 0.75 % Triton X-100 (v/v) in PBS (2 h, RT). Subsequently, slices were incubated with respective antibodies (anti-GFP, ab6556, 1:1000, abcam; anti-PDGF-receptor α, AF1062, 5 µg/ml, R&D-Systems; in PBS, 5 % BSA, 0.025 % Triton X-100; overnight at 4 °C) to detect cGi-500 and identify cardiac fibroblasts^{42 (link)}, respectively. After washing (3x PBS, 5 min, RT), slices were incubated with secondary antibodies (Alexa488 donkey anti-rabbit, A21206, Thermo Fisher, 1:1000; Alexa594 donkey anti-goat, A11058, Thermo Fisher, 1:1000; in PBS, 5% BSA, 0.025% Triton X-100; 2 h, RT, dark). After washing (3x PBS, 5 min, RT), the slices were covered in aqueous embedding solution (Mount FluorCare DAPI, Carl Roth, Karlsruhe, Germany) and analysed by confocal laser scanning microscopy (Nikon Eclipse Ti-E Inverted Microscope System). Laser and microscope settings were kept identical for different genotypes.