Mouse anti ubx

Embryos were collected on grape agar plates, washed into mesh baskets, and dechorinated in 50% bleach for 2 min. Embryos were then devitellenized and fixed in a 1:1 mixture of methanol–heptane before storing in methanol at −20°C. Embryos were gradually rehydrated in a series of increasing PTx (PBS with 0.1% Triton):methanol mixtures (1:3, 1:1, and 3:1) before washing for 5 min in PTx. Embryos were then blocked in PTx supplemented with 5% normal donkey serum and 0.2% azide for 1 h at room temperature. Blocked embryos were then incubated with the primary antibody overnight at 4°C. The following primary antibodies were used: rabbit anti-Wde (1:500; A. Wodarz), rabbit anti-H3K9me3 (1:500), mouse anti-Ubx (1:20; Developmental Studies Hybridoma Bank [DHSB], FP3.38), and mouse anti-HP1a (1:100; DSHB, CA19). Embryos were then washed three times with PTx for 15 min each and then incubated with the appropriate fluorescently labeled secondary antibody (Molecular Probes) at 1:500 for 1 h in the dark at room temperature. Embryos were then washed again three times with PTx for 15 min each. DAPI was added to the second wash. Finally, stained embryos were mounted on glass slides in Fluoromount.