Tube Staining Method for Oocyte Identification

The tube staining method was performed on dissected gonads fixed in 3% paraformaldehyde and methanol (Chen and Arur, 2017 (link)). The samples are washed using 1X PBST (0.1% tween), blocked with 30% NGS and incubated with primary antibodies at 4°C overnight. Appropriate secondary antibodies were added and incubated at room temperature for 1–2 h followed by three washes with 1X PBST with DAPI included in the final wash and samples were mounted on a 3% agar pad with Vectashield mounting medium. The primary antibodies used in this study are: Rat monoclonal OLLAS epitope tag antibody (1:200, Novus Biologicals, Cat. No. NBP1-06713) and Rabbit anti-phospho-Histone H3 (Ser10) antibody (1:200, EMD Millipore Cat. No. 06–570). Secondary antibodies were goat-anti-rat Alexa Fluor 568 nm and goat-anti-rabbit Alexa Fluor 488 (1:1,000, Invitrogen). pH3 was used as a control of the staining protocol allowing us to identify mature oocytes.