Biotinylated donkey anti goat igg

Foetal heart sections were subjected to antigen retrieval in citric acid buffer (0.01 M, pH 6.0) for 12 minutes at 94°C in a microwave (BP‐111, Microwave Research & Applications). E18.5 heart sections were then stained using anti‐α‐smooth muscle actin primary antibody (1:3000; Sigma), biotinylated griffonia simplicifolia lectin‐1 (1:200; Vector Laboratories) and anti‐sex‐determining region Y protein antibody (1:200, Santa Cruz) to visualize coronary arteries, capillaries and Y chromosome, respectively. To analyse proliferation and epicardial epithelial‐to‐mesenchymal transition (EMT), E12.5 heart sections were incubated in anti‐phosphorylated histone H3 (pHH3, 1:500; Abcam) and anti‐Wilm's tumor‐1 (Wt1, 1:300; Calbiochem) primary antibodies, respectively. Primary antibodies were left on overnight at room temperature in humidity chambers. Sections were subsequently incubated for one hour at room temperature with secondary antibodies; biotinylated donkey anti‐goat IgG (1:500), biotinylated goat anti‐rabbit IgG (1:500) or biotinylated goat anti‐mouse IgG (1:500) (Vector Laboratories). Slides were imaged using the Zeiss Observer D1 microscope and AxioVision Rel 4.7 Software. All quantifications of histological images were performed using ZEN microscope software (Zeiss).13, 15, 17

For single immunoperoxidase staining for either CTB, ChAT or CSPG selected brainstem sections were processed free-floating with polyclonal goat antibodies directed against either CTB (1:20,000; List) or ChAT (1:100; Chemicon) or with rabbit anti-CSPG (1:5000; Biogenesis) for 48 h at 4°C. All markers were visualized by binding of biotinylated donkey-anti-goat IgG (1:200; Vector Lab) or biotinylated donkey anti-rabbit IgG (1:200; Vector Lab) followed by extravidin-peroxidase (1:1000; Sigma) and diaminobenzidine (DAB) as chromogen to yield a brown color.