Rabbit anti piwi

Ovaries were dissected in cold PBS and fixed in PBS with 4% paraformaldehyde for 15 min, then washed with PBT (PBS and 0.3% Triton X-100) five times for 15 min each. The ovaries were incubated in 0.5% goat serum diluted in PBT for 1 hr. Appropriate primary antibodies were added to PBS and incubated at 4° overnight, then washed with PBT five times for 15 min each. Appropriate secondary antibodies were then added and incubated at 25° for 2 hr; they were washed with PBT five times for 15 min each. After the last wash, the stained ovaries were mounted in Fluoromount mounting media (F4680; Sigma). Images were obtained with an inverted Zeiss LSM780 fitted with a UV laser.
The following primary and secondary antibodies were used: mouse monoclonal anti-Hts antibody 1B1 (DSHB, 1:100); rabbit anti-Piwi (1:200; Santa Cruz sc-98264); FITC-conjugated anti-mouse IgG (1:200; Jackson ImmunoResearch); and TRITC-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch).

Ovaries from young females were dissected and fixed in PBS, 4% formaldehyde, 0.1% Triton-X100 for 20 min, washed in PBS with 0.1% Triton-X100. The primary antibody dilutions for immunostaining were 1:200 for rabbit anti-Piwi (Santa Cruz-98264), 1:8000 for guinea pig anti-tj (gift from D. Godt, Toronto) and 1:1000 for rabbit anti-dcr2 (gift from F. Gebauer, Barcelona) antibodies. The anti-rabbit and anti-guinea pig secondary antibodies were 1:400 and 1:200 diluted, respectively. Ovaries were Dapi stained and mounted in Vectashield medium (Vector Laboratories). Fluorescent images were acquired with a Zeiss Apotome microscope. Images were treated in Omero (https://www.openmicroscopy.org/omero/).