Gonads were dissected from 1-day adults of him-17::3xHA worms in 1X sperm salts (50 mM PIPES pH 7.0, 25 mM KCl, 1 mM MgSO4, 45 mM NaCl, and 2 mM CaCl2) with 1 mM levamisole and fixed in 1% paraformaldehyde diluted in 1X sperm salts for 5 min in a humid chamber. Slides were then frozen on a metal block on dry ice for at least 10 min prior to flicking off the coverslip and immersing in 100% ethanol at −20° C for 2 min. Slides were then washed in PBSTB [1XPBS with 0.1% Tween and 0.1% bovine serum albumin (BSA)], and incubated overnight at 4°C with primary antibody: mouse anti-HA (Cell Signaling), guinea pig anti-XND-1(Wagner et al., 2010 (link)), and rabbit anti-HTP-3 (Das et al., 2022 (link)) (all diluted 1:2000 in PBST). The next day, slides were washed 3X in PBSTB for 10 min and incubated with secondary antibodies: anti-mouse Alexa 488 (Molecular Probes), anti-guinea pig 633, and goat anti-rabbit Alexa 568 (Invitrogen, all diluted 1:2000 in PBSTB) for 2 h at room temperature in the dark. Slides were then washed 2 X 10 min in PBSTB, mounted in Prolong Gold without DAPI (Invitrogen) and put in the dark to cure overnight before imaging. The images were taken using a Stellaris8 TauSTED equipped with three depletion lines.
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