Wild-type embryos were collected for 1 h and matured for 3 h at 25°C, then dechorionated with 50% household bleach for 3 minutes and fixed for 20 minutes with 1:4 (v/v) solution of 10% formaldehyde (Polysciences, Warrington, PA, USA) and heptane (Sigma, St. Louis, MO, USA). Fixed embryos were devitellinized by shaking vigorously in 1:1 methanol/heptane, after which they were washed three times with methanol and once with 100% ethanol, and stored in ethanol at -20°C.
Embryos were rehydrated in phosphate buffered saline pH 7.2, 0.05% Tween20, 0.2% TritonX-100 (PBT+Tx), post-fixed for 20 minutes in 5% formaldehyde/PBT+Tx, and, after several washes in hybridization buffer (50% formamide, 5 × SSC pH 5.2 to 5.4, 0.2% TritonX-100, 50 μg/ml heparin) at 55 to 59°C, prehybridized for 1 to 5 h in hybridization buffer. There was no proteinase K treatment. To improve the staining quality, the prehybridized eggs were stored in -20°C hybridization buffer for at least 16 h.
For each in situ hybridization, 50 to 100 μl of embryos were incubated in 300 μl of hybridization buffer with an RNA probe for one gene labeled with DIG and an RNA probe to a second gene labeled with either DNP or BIO. After 12 to 48 h co-hybridization at 55 to 59°C and several high-stringency and low stringency washes, the two probes were detected sequentially. The DIG-labeled probe was detected using 1:500 horseradish peroxidase conjugated anti-DIG-antibody (anti-DIG-POD; Roche, Basil, Switzerland) and either a Cy3 or coumarin-tyramide reagent (Perkin-Elmer TSA-kit, Wellesley, MA, USA). Before the second probe was detected, the anti-DIG-POD antibody was first removed with several 15 minute washes with 50% formamide, 5 × SSC, 0.2% TritonX-100 at 55°C, followed by inactivation of the remnants with 5% formaldehyde/PBT+Tx. Then the second probe was detected using 1:100 anti-DNP-HRP (Perkin-Elmer) and either the complementary coumarin or Cy3-TSA-tyramide reaction. To allow detection of nuclei with a nucleic acid binding stain, all RNA in the embryo was first removed by digestion with 0.18 μg/ml RNAseA in 500 μl overnight at 37°C, and then the DNA was stained overnight by incubation in 500 to 1,000 μl of a 1:5,000 dilution of Sytox Green dye (Molecular Probes, Carlsbad, CA, USA).
The kni protein expression was detected with guinea pig-anti-kni (a gift from J Reinitz, Stony Brook University, Stony Brook, NY, USA) and Alexa488-anti-guinea pig (Molecular Probes) in embryos hybridized against ftz DIG-mRNA that was detected with coumarin tyramides. For these embryos only, the nuclei were detected using mouse-anti-histoneH1 and Alexa555-anti-mouse.
The stained embryos were dehydrated with an ethanol-series and mounted in xylene-based DePex (Electron Microscopy Sciences, Hatfield, PA, USA). A #1 coverslip was placed on a bridge formed by two #1 coverslips to prevent embryo flattening. This mountant has the advantages of creating permanent slides that protect the fluorophore from oxygen, which makes the samples highly resistant to photobleaching. To estimate the refractive index of the mountant (which determines the scaling of the z-axis), we used the assumption that embryo morphology was independent of the orientation of the embryo when it was imaged. A d/v cross-section of multiple embryos was taken at 50% egg length. Within these cross-sections, the ratio of the d/v length to the left/right length was plotted against orientation angle (data not shown). The refractive index was then computed so that this ratio was independent of the orientation. The average refractive index calculated using this method was 1.62 ± 0.06.
Embryos were rehydrated in phosphate buffered saline pH 7.2, 0.05% Tween20, 0.2% TritonX-100 (PBT+Tx), post-fixed for 20 minutes in 5% formaldehyde/PBT+Tx, and, after several washes in hybridization buffer (50% formamide, 5 × SSC pH 5.2 to 5.4, 0.2% TritonX-100, 50 μg/ml heparin) at 55 to 59°C, prehybridized for 1 to 5 h in hybridization buffer. There was no proteinase K treatment. To improve the staining quality, the prehybridized eggs were stored in -20°C hybridization buffer for at least 16 h.
For each in situ hybridization, 50 to 100 μl of embryos were incubated in 300 μl of hybridization buffer with an RNA probe for one gene labeled with DIG and an RNA probe to a second gene labeled with either DNP or BIO. After 12 to 48 h co-hybridization at 55 to 59°C and several high-stringency and low stringency washes, the two probes were detected sequentially. The DIG-labeled probe was detected using 1:500 horseradish peroxidase conjugated anti-DIG-antibody (anti-DIG-POD; Roche, Basil, Switzerland) and either a Cy3 or coumarin-tyramide reagent (Perkin-Elmer TSA-kit, Wellesley, MA, USA). Before the second probe was detected, the anti-DIG-POD antibody was first removed with several 15 minute washes with 50% formamide, 5 × SSC, 0.2% TritonX-100 at 55°C, followed by inactivation of the remnants with 5% formaldehyde/PBT+Tx. Then the second probe was detected using 1:100 anti-DNP-HRP (Perkin-Elmer) and either the complementary coumarin or Cy3-TSA-tyramide reaction. To allow detection of nuclei with a nucleic acid binding stain, all RNA in the embryo was first removed by digestion with 0.18 μg/ml RNAseA in 500 μl overnight at 37°C, and then the DNA was stained overnight by incubation in 500 to 1,000 μl of a 1:5,000 dilution of Sytox Green dye (Molecular Probes, Carlsbad, CA, USA).
The kni protein expression was detected with guinea pig-anti-kni (a gift from J Reinitz, Stony Brook University, Stony Brook, NY, USA) and Alexa488-anti-guinea pig (Molecular Probes) in embryos hybridized against ftz DIG-mRNA that was detected with coumarin tyramides. For these embryos only, the nuclei were detected using mouse-anti-histoneH1 and Alexa555-anti-mouse.
The stained embryos were dehydrated with an ethanol-series and mounted in xylene-based DePex (Electron Microscopy Sciences, Hatfield, PA, USA). A #1 coverslip was placed on a bridge formed by two #1 coverslips to prevent embryo flattening. This mountant has the advantages of creating permanent slides that protect the fluorophore from oxygen, which makes the samples highly resistant to photobleaching. To estimate the refractive index of the mountant (which determines the scaling of the z-axis), we used the assumption that embryo morphology was independent of the orientation of the embryo when it was imaged. A d/v cross-section of multiple embryos was taken at 50% egg length. Within these cross-sections, the ratio of the d/v length to the left/right length was plotted against orientation angle (data not shown). The refractive index was then computed so that this ratio was independent of the orientation. The average refractive index calculated using this method was 1.62 ± 0.06.
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