3 3 diaminobenzidintetrahydrochloride

Tissue sample cross-sections (4 µm) mounted on Superfrost Plus slides were air dried and incubated at 60° C for 30 min. For immunostaining, sections were deparaffinized twice in chloroform for 15 min following rehydratization in ethanol solutions with decreasing concentrations (100%, 99%, 96%, 90%, 70%, and 50%), each for 2 min. Thereafter, endogenous peroxidase activity was quenched light-proof with 3% H₂O₂ in PBS-T for 15 min at room temperature. Antigen presentation was performed in 0.1 M citrate buffer for 15 min at 100° C. An immunostaining procedure was then performed on the sections at room temperature. Non-specific binding sites were blocked with 10% goat serum (Dako Deutschland GmbH, Hamburg, Germany) in PBS-T for 30 min. A specific antibody raised in rabbits against FGF1 [23 (link)] and anti-FGF2 [24 (link)] kindly provided by Dieter Schams (Physiology Weihenstephan, Freising, Germany) was diluted 1:50 in PBS-T and applied overnight at 4 °C. The samples were washed in PBS-T and incubated with HRP-conjugate secondary antibody (goat anti-rabbit, Sigma-Aldrich Chemie GmbH, München, Germany) for 1h. Immunostaining was performed using 3,3′-diaminobenzidintetrahydrochloride (Sigma-Aldrich Chemie GmbH, München, Germany) in 0.3% H₂O₂ PBS for 2 to 10 min. The tissue samples were counterstained with Mayer’s Haemalaun (Carl Roth GmbH, Karlsruhe, Germany).

Heart morphology was analyzed in postnatal day 0 (P0) mice and cell proliferation was analyzed by phospho-histon H3 (pHH3) staining in E12.5 hearts. Briefly, the mouse thorax was fixed in 4% paraformaldehyde overnight, dehydrated in ethanol, embedded in paraffin and serially sectioned into 5-μm sections. Heart sections were stained with hematoxylin/eosin (H/E) and images were captured using a light microscope (Observer D1, Zeiss, Germany). Images were taken on every 25 μm of the heart and the three-dimensional visualization of heart structures was reconstructed using AMIRA® program. To analyze cell proliferation and apoptosis, heart sections were immunostained using anti-pHH3 (phospho S10) antibody (Abcam) and anti-cleaved claspase-3 antibody (Cell Signaling), respectively, followed by incubation with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA). Signals were visualized by 3-3′di-aminobenzidin tetrahydrochloride (Sigma-Aldrich Chemie, St. Louis, MO, USA). Counterstaining was performed with modified Mayer’s hematoxylin (Thermo Scientific, Waltham, MA, USA). The number of pHH3⁺ cells from at least 3 individual heart sections per sample was quantified and normalized to areas of the myocardium.