Fluorophore conjugated goat anti rabbit

Immunofluorescence was performed on 10 μm frozen sections. In brief, sections were dried at room temperature for 20 minutes and then washed 3 times in 1x PBS. Sections were incubated in blocking solution containing 5% normal goat serum and 3% bovine serum albumin in PBS for 60 minutes at room temperature. Rabbit anti-activated caspase 3 antibody (1:100, Cell Signaling Technologies, Inc.) or rabbit antibody against phospho histone H3 (1:300, Millipore) and rat antibody against pan-cytokeratin (1:300, Developmental Studies Hybridoma Bank) were applied to samples and incubated overnight at 4 °C. Sections were next washed in PBS three times and incubated with fluorophore-conjugated goat anti-rabbit (1:600, for activated caspase 3) or donkey anti-rat (1:800, for pan-cytokeratin) IgG (Invitrogen, Inc.) for 45 minutes at room temperature. Samples were then washed in PBS 5 times and covered with DAPI-containing Vectasheld mounting medium (Vector Laboratories, Inc.). Immunofluorescent signal for pan-cytokeratin marked the Wolffian duct and weakly labeled the Müllerian duct at E12.5 (Orvis and Behringer, 2007 (link)). Cytokeratin immunofluorescence and DAPI signal were both used to distinguish Müllerian duct from nearby structures and for MDE cell counting.