Sc 136083

Approximate midsections of d4, d8 and d16 ovaries were dewaxed in Histochoice solvent (Sigma) and re-hydrated in changes of ethanol of decreasing concentrations. Slides were immersed in 0.01 M citrate buffer (pH 6.0) and microwaved for 4 × 5 min to retrieve antigens before washing in phosphate buffered saline (PBS; pH 7.4). Non-specific binding was blocked with CAS-Block (ThermoFisher) for 20 min before applying a mixture of rabbit anti-SMAD2/3 (1:400; #5678; Cell Signalling Technology) and mouse anti-STRAP (0.3 μg/mL; sc-136083; Santa Cruz Biotechnology) diluted in CAS-Block overnight at 4°C. Primary antibodies were replaced on some sections with equivalent concentrations of non-immune mouse IgG (Vector Laboratories, Bakewell, UK) or rabbit IgG (Vector) to determine non-specific binding. After washes in PBS, all sections were incubated at room temperature for 45 min in a mixture of Alexa555 anti-mouse IgG (1:400; Invitrogen) and Alexa488 anti-rabbit IgG (1:400; Invitrogen) diluted in PBS. Sections were washed in PBS and mounted in ProLong Gold antifade reagent with DAPI (Invitrogen) and imaged using a Leica inverted SP5 confocal laser scanning microscope (Leica Microsystems). Images presented in this study were taken from sections stained in the same run, using the same laser and gain settings.