Alexa 488 568 conjugated donkey anti mouse rabbit secondary antibodies

40 μm sections containing the mHb from ChAT-Cre::tdTom mice were washed three times for 10 min in 0.1M PBS (pH 7.4). Subsequently, sections were incubated in 10% normal donkey serum (NDS) with 0.5% Triton X-100 in PBS for 30 min at RT. The sections were then incubated with the Tcf7l2 primary antibody (#17–10109, Millipore Sigma, MA, USA) in 10% NDS, 0.5% Triton X100 in PBS overnight at 4°C. Slides were then washed 4× 10 min with 0.1 M PBS. Immunoreactivity was probed using Alexa 488/568-conjugated donkey anti-mouse/rabbit secondary antibodies (1:1000; Molecular Probes, OR, USA) for 1 h at room temperature. The secondary antibodies were diluted in PBS-T containing 2% NDS. The slides were mounted with Fluoro-Gel containing DAPI (Electron Microscopy Sciences, PA, USA). The images were collected using a Zeiss AxioImager Z2 microscope system.