FISH was performed as previously described20 (link) using oligonucleotides 20 nt in length and complementary to transcripts nos-RA (CG5637; 75 probes), cycB-RA (CG3510; 48 probes), pgc-RA (CG32885; 48 probes), and osk-RA (CG10901; 99 probes). Sequences are listed in Supplementary Table 1 . Custom oligonucleotides with 3′ amine modification were obtained from Biosearch Technologies, conjugated to either Atto 565 (Sigma 72464) or Atto 633 (Sigma 43429) dye, and purified by HPLC as previously described51 (link). Intact ovaries were dissected from well-fed 2–4 day old females and processed for FISH as described23 (link). Imaging was performed using a 63x HCX PL APO CS 1.4 NA oil immersion objective on a Leica TCS SP5 laser-scanning confocal microscope equipped with GaAsP “HyD” detectors in photon counting mode, with pixels of 76 x 76 nm and z spacing of 340 nm. For each probe set, laser power was adjusted to optimize separation of signal and noise. The same settings were used repeatedly for all samples treated with a given probe set. For each experiment, image stacks were acquired from 3–5 different egg chambers/oocytes or embryos at each developmental stage shown. Each stack contained 20,000–100,000 particles, depending on the volume imaged and the size of the egg chamber/oocyte/embryo. Laser power exhibited fluctuations of ~10% between experiments; however, normalization to single mRNA intensity nullifies any effect in our measurements of absolute mRNA amount. For egg chambers/oocytes, z-series represent approximately half the thickness of the tissue starting from near the interface between the follicle cells and the nurse cells/oocyte. For embryos, image stacks extend from the cortical surface to near the midsagittal plane.
As controls for goodness of single particle detection, we compared the densities of punctae in early embryos from wild-type females and from females heterozygous or homozygous for the maternal RNA-null nosBN allele. As expected, heterozygous embryos contain half as many particles as in wild-type (0.39 ± 0.01 versus 0.75 ± 0.02 particles/μm3, n = 4 embryos each). In homozygous nosBN embryos, the number of particles/volume exceeding the “difference of Gaussians” detection threshold52 (link) was <1% of the number detected in wild-type. Similarly for osk, embryos from females heterozygous for the mRNA-null allele oskA87 contain half the number of particles of wild-type embryos (0.24 ± 0.03 versus 0.41 ± 0.03 particles/μm3 in wild-type, n = 4 embryos each). These results are similar to the two-fold reduction in particle number observed for maternally supplied hunchback (hb) mRNA in hb deficiency heterozygotes20 (link) and support previous findings20 (link),21 (link) that the FISH method effectively distinguishes true objects from imaging noise.
As controls for goodness of single particle detection, we compared the densities of punctae in early embryos from wild-type females and from females heterozygous or homozygous for the maternal RNA-null nosBN allele. As expected, heterozygous embryos contain half as many particles as in wild-type (0.39 ± 0.01 versus 0.75 ± 0.02 particles/μm3, n = 4 embryos each). In homozygous nosBN embryos, the number of particles/volume exceeding the “difference of Gaussians” detection threshold52 (link) was <1% of the number detected in wild-type. Similarly for osk, embryos from females heterozygous for the mRNA-null allele oskA87 contain half the number of particles of wild-type embryos (0.24 ± 0.03 versus 0.41 ± 0.03 particles/μm3 in wild-type, n = 4 embryos each). These results are similar to the two-fold reduction in particle number observed for maternally supplied hunchback (hb) mRNA in hb deficiency heterozygotes20 (link) and support previous findings20 (link),21 (link) that the FISH method effectively distinguishes true objects from imaging noise.
