Histology and Embryology

2.4.1 Formaldehyde fixation of Drosophila embryos

For fixation, embryos were collected and dechorionated as described in section 2.3.1. Collection intervals of 2-4h embryonic age were

Materials and methods

chosen. For stainings using the anti-Vasa antibody, the embryos then were fixated on the shaker in 8ml Heptane, 0,5ml Formaldehyde (37%) and Fixation buffer (100 mM HEPES; 2 mM MgSO4; 1 mM EGTA, pH 6,9) for 20’ (modified from Rothwell and Sullivan, 2000). The aqueous phase was removed. The embryos were devitellinized by addition of methanol and vigorous shaking. The Heptane- and interphase were removed and the embryos were three times washed with methanol.

For storage, the embryos in methanol were kept at –20°C.

2.4.2 Paraformaldehyde fixation of embryos

For stainings of epithelial morphology, embryos were fixated on the shaker in 5ml Heptane, in 4ml PEM-buffer (100 mM PIPES, 5 mM EGTA, 2 mM MgCl2, pH 6.8, 0.2% Triton X-100 (TX-100)) including 5% PFA and 1 ml Picric acid (Sigma; 13% aqueous solution) for 20’ (modified from Kreitmeier et al., 1995). The aqueous phase was removed. The embryos were devitellinized by addition of methanol and vigorous shaking. The Heptane- and interphase were removed and the embryos were three times washed with methanol. For storage, the embryos in methanol were kept at –20°C.

For stainings using fluorescently labeled phalloidin, fixation was carried out as described above replacing Methanol with 80% ethanol.

2.4.3 Dissection and fixation of ovaries

Ovaries were dissected as described in section 2.3.2 and fixated in 5ml PEM-PFA (100 mM PIPES, 5 mM EGTA, 2 mM MgCl2, pH 6.8, 0.2% Triton X-100 (TX-100)) including 4% PFA for 20 min. Fixation was stopped by 2x rinsing with PBT (PBS, 0,1% Tween20). For storage, ovaries were dehydrated stepwise in ethanol and stored at -20°C.

Materials and methods

2.4.4 Generation of transcript specific probes for RNA in situ detection For labeling of RNA-probes, the RNA- Labeling and Detection Kit (Roche) was used according to the manufacturers manual. After labeling, 1 µl DNaseI (RNase-free) was added to the samples followed by 15 min incubation at 37°C. The labeled probe was purified using the RNeasy Mini Kit (QIAGEN), following the instruction manual.

2.4.5 RNA in situ detection in fixated embryos and ovaries

For RNA in situ staining (modified from Lehmann and Tautz, 1994) ovaries were dissected and fixated as described in section 2.4.3. The ovaries were washed in PBT 3 x 5 min and prefixated for 20 min in PBS including 4% formaldehyd. After further washing steps with PBT, the ovaries were transferred into the hybridization solution in a stepwise manner. Equilibration steps included 10 min in (1:1) PBT:Hype-B (50 % Formamid; 5 x SSC; 0,1 % Tween20), in 10 min Hybe-B and 10 min in (1:1) Hybe-B:HybeA (50% formamide, 5x SSC, 0.2 mg/ml sonicated salmon testis DNA, 0.1 mg/ml tRNA, 50 µg/ml heparin).

The ovaries were prehybridized in Hybe-A for 1h at 65°C. Then, the buffer was replaced by 30 µl Hybe-A including 2 µl DIG-labeled RNA-probe. The ovaries were incubated over night at 65°C in a waterbath.

After hybridization, the ovaries were washed 10 min at 65°C with prewarmed Hybe-A, 2 x 15 min at 65°C with prewarmed Hype-B, 15 min at RT in PBT:Hybe-B (1:1) and 4 x 15 min at RT in PBT. For detection of the DIG-labeled RNA-Probes, the ovaries were incubated with an anti-Dig AP-conjugated antibody (1:2000, Roche Diagnostics, Mannheim) for 1h at 37°C. After 6 x 15 min washing in PBT at RT the ovaries were transferred into AP-Puffer (20 mM Tris pH 9,5; 100 mM NaCl; 50 mM MgCl2) and washed 3 x 5 min. For the development of the staining, 4,5 l NBT and 3,5 l BCIP were added into 1 ml AP-Puffer.

The staining was stopped by 3 x 5 min and 1 x 20 min washing in PBT.

Materials and methods

The stained ovaries were dehydrated in an ethanol dilution series (30%, 50%, 70%, 90% and 2 x 100%), mounted on a slide in Canada Balsam (Sigma-Aldrich) and covered with a coverslip.

2.4.6 Antibody staining of Drosophila embryos

About 20 µl of fixated embryos were rehydrated by three times 5’

washing with PBT including 5 % goat serum (protocol adapted from Rothwell and Sullivan, 2000). Incubation with the primary Antibody was done at 4°C over night. The primary antibody was then removed by twice washing with PBT including 2% of goat serum. Then, either fluorescence coupled or biotinylated secondary antibodies (Molecular Probes) were applied, diluted in 380 µl PBT including 10 µl Serum, and incubated on the embryos for 2 h. All following steps were carried out at RT.

For detection of the fluorescence-coupled antibodies, the embryos after incubation were washed 6 x 10‘ in PBT, 2 x 10‘ in PBT and 2 x 5‘ in PBS. The embryos were allowed to sediment at the tip of the reaction vial, taken up, mounted on a slide with ProLong Gold antifade reagent (Molecular Probes) and covered with a coverslip and sealed with custom nail polish.

The non-bound biotinylated Antibodies were removed by 2 x 5’

washing with PBT. For the coupling of Horseradish peroxidase (HRP) to the biotinylated secondary antibodies, the ABC-Kit Vectastain (Vector Laboratories) was used according to the manufacturers instruction. For HRP detection, the embryos were transferred into 500 µl PBT including 10µl Diamino-Benzidine (10 mg/ml in 50 mM Tris-HCl; Sigma) and 10 µl hydrogenperoxide (0,3% in PBT; Fluka). The embryos were then stepwise dehydrated (2 x 10’ in Ethanol 70% and 2 x 10’ in Ethanol 100%), mounted on a slide with Canada balsam (Sigma), covered with a coverslip and sealed with custom nailpolish.

Materials and methods

2.4.7 Microscope / Confocal imaging

For taking Confocal images of embryos and cells, the Leica DMRXA2 Confocal Microscope (Leica Microsystems) was used. For image processing, the softwares ImageJ (National Institutes of Health) and Adobe Photoshop (Adobe Systems) were used.

2.4.8 In vivo imaging of embryonic development

For time lapse imaging of germ cell migration, 2 h collections of embryos were made. The embryos were arranged on an apple agar slice and fixed to a coverslip with embryo glue and covered with Halocarbon-Oil (Atochem) (protocol modified from germ line transfection protocol, section 2.2.1; Rubin and Spradling 1982). The coverslip was then applied on an Aluminum slide with a notch fitting the hanging drop with the embryos. Images of the developing embryos were taken from 1to 12 hours.

For the measurement of cellularization speed, 30 min collections of embryos were treated as described above. For analysis, the Axiovert 200M (Leica) was used, which was operated with the software Openlab (Improvision). Data analysis was conducted with the software ImageJ (National Institutes of Health).

2.4.9 Cuticle preparations

12 h embryo collections were aged in order to allow for complete hatching of viable embryos. The unhatched embryos were dechorionated, mounted in Hoyers Lactate medium (30 g gum Arabic, H2O to 50 ml, 200 g chloral hydrate 20 g Glycerol, 150 ml lactic acid) (modified from Stern and Sucena, 2000) and incubated at 60°C overnight.

Materials and methods