Immunofluorescence and FISH Protocols for Drosophila Embryos and Ovaries

Immunofluorescence and FISH were performed on embryos and ovaries as described in references [4] (link), [83] (link). Polytene chromosomes were dissected in 0.7% NaCl, squashed, and fixed in 1.8% PFA, 45% acetic acid for 17 minutes. They were then washed in 1% Triton X in PBS for 10 minutes, then washed in 5% milk in PBS for 1 hour, incubated with primary antibody overnight at 4°C, washed in 5% milk in PBS for 10 minutes, incubated with secondary antibody for 1 hour at room temperature, and then washed for 10 minutes in buffer A (0.15M NaCl, 0.2% NP40 substitute, 0.2%Tween 20) followed by 10 minutes in buffer B (0.20M NaCl, 0.2% NP40 substitute, 0.2%Tween 20).
Rat anti-HA antibody (Roche, 3F10) was used at 1∶100, rat anti-Vasa (DSHB) was used at 1∶25, Fibrillarin (Abcam, Ab5281) was used at 1∶100, anti-HP1a antibody (C1A9, DSHB) was used at 1∶100. Alexa fluorophore-conjugated secondary antibodies were used to detect the primary antibody. Fluorescently labeled probes against GA-rich satellites, AACAC, 2L3L, 359 bp and dodeca were obtained from Sigma with sequences described in references [8] (link), [83] (link), [97] . Imaging was carried out using a Zeiss 710 confocal microscope at Cornell University's Microscopy and Imaging Facility.