MATERIALS AND METHODS

Fly stocks
Sciara (Bradysia) coprophila stock 7298 (wild type with wavy wing marker linked to the X' inversion chromosome) and 7298 derivative stocks (“Holo”) were raised in the laboratory at 21°C. Only female fourth instar larvae from these stocks were used for immunofluorescence studies because they undergo an additional endoreduplication cycle (Rasch 1970), resulting in larger polytene chromosomes. Well-fed larvae grown in medium density conditions yielded chromosome squashes of significantly better quality. The expansion of puff morphology coincides with development of the eye anlage from mid to late fourth larval instar, allowing larvae to be staged by counting their eyespot granules under a 16X objective. Each developmental stage is specified by the number of eyespot granules in the longest row multiplied by the number of rows minus one (Gabrusewycz-Garcia 1964, Wu et al. 1993). At DNA puff II/9A, amplification initiates at 10 x 5 eyespot stage and continues through 12 x 6, while maximal puffing resulting from a burst of transcription occurs at the 14 x 7 stage (Gerbi et al. 1993, Wu et al. 1993, Foulk et al. 2006).

Antibodies
Isoform A of the ecdysone receptor (EcR-A) predominates in the salivary glands of fourth larval instar Sciara coprophila (Foulk et al. in prep). A polyclonal antibody to this isoform was produced against the unique EcR-A 5' nucleotide sequence as described elsewhere (Foulk et al. in prep). The antibody preparation was then affinity purified with Affi-Gel® 10 Activated Affinity Media (BioRad, Hercules, Calif.) coupled to the EcR-A specific protein fragment, and the specificity of this antibody was checked by immunoblotting. Alexa Fluor 488 dye-conjugated goat anti-rabbit IgG was obtained from Molecular Probes, Invitrogen (Eugene, Oregon).

Preparation of chromosome squashes and immunofluorescent detection of EcR-A
Polytene chromosome squashes and immunostaining were performed as described by Zink et al. (1991) with modifications. Briefly, the anterior portions of salivary glands were excised from staged larvae in dissecting buffer (PBS pH 7.4, 0.1% Triton X-100) [PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4] and immediately fixed for 25 s in freshly prepared solution I (3.7% formaldehyde, 1% Triton X-100, PBS pH 7.4). Glands were then transferred to a 35 μl drop of solution II (3.7% formaldehyde, 50% acetic acid) on a siliconized coverslip for 70 s. The 37% formaldehyde stock solution used for solutions I and II was made fresh before each experiment by boiling paraformaldehyde (Sigma, St. Louis, Missouri) in 15 mM KOH until dissolved. After picking up the coverslip with a poly-L-lysine coated slide, the entire preparation was flipped over [slide coating: clean slides were soaked for 5 min in 0.1% poly-L-lysine (Sigma, St. Louis, Missouri) and oven-dried at 80°C for 1 h]. Chromosomes were spread by firmly tapping the coverslip around the glands with the eraser end of a pencil. The chromosomes were then flattened by compression of the slide between several sheets of blotting paper with thumb pressure. After freezing the preparation in liquid nitrogen, the coverslip was pried off with a razor blade and the exposed chromosome squash was washed in PBS (2 x 15 min, agitating). Slides were then blocked (PBS pH 7.4, 3% nonfat dried milk, 0.3% Triton X100) for 1 h at room temperature or overnight at 4°C, and washed (2 x 10 min, agitating) with PBT (PBS pH 7.4, 0.1% BSA, 0.2% Tween 20).

Chromosome preparations were reacted sequentially with anti-EcR-A (1:2500 in PBT) and then Alexa Fluor 488 dye-conjugated goat anti-rabbit IgG (1:1000 in PBT) for 1 h each. Each immunostaining step was followed by a PBT wash (2 x 10 min, agitating). Slides were then counterstained with DAPI (200 ng/ml in H2O) for 10 min. All incubation steps were carried out at room temperature in a darkened humid chamber. After a brief rinse in PBT, slides were mounted with ProLong® Gold (Invitrogen, Carlsbad, Calif.) and stored in the dark at 4°C. Reacted preparations were examined under a Carl Zeiss Axiovert 200 M microscope and photographed with the 40X objective (dry). The accompanying AxioVision software was used to optimize exposure times automatically according to the immunostaining intensity of each chromosome preparation. Captured images were then exported into Adobe Photoshop® for resizing and cropping.