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In Situ Hybridisation with DIG-RNA Probes On Embryos (Ilan Davis, July 1996) 1. Collect embryos on apple juice / grape juice plates and remove them with water and a paint brush to a sieve. Rinse well with water to remove yeast. Try to avoid including dead flies and lumps of agar. Dechorionate in house-hold bleach diluted 1:1 with tap water for 2 minutes. Rinse thoroughly in tap water or embryo wash (0.05% Triton or Tween20, 0.7% NaCl) to stop eggs sticking to sides of sieve. Remove excess water (not completely dry, as embryos stick to sieves). 2. Fixation: (two alternate methods) a. For best microtubule preservation: Transfer the embryos from the sieve into 10ml heptane in a universal bottle with well sealed lids. After 30 seconds add 10ml of 37% formaldehyde (stock solution) and agitate gently for 5 minutes. DO NOT SHAKE VIGOROUSLY. b. More conventional fixation: Transfer the embryos from the sieve into equal volumes of heptane and freshly prepared 3.7% formaldehyde in 1x PBS (1:10 dilution of stock formaldehyde solution) in a universal bottle and fix for 15-20 minutes, initially shaking vigorously. 3. Devitellinisation: Remove lower phase (fix) and half of upper phase (heptane) add MeOH almost to the top of the tube. Shake vigorously. Note that there should still be two phases, if not add some extra heptane, as there must be two phases for the devitellinisation to work. Devitellinised embryos sink to the bottom of the Lower MeOH phase within 1-2 minutes and can be collected with a glass Pasteur pipette. Embryos remaining at the interface should be discarded. Rinse x2 with MeOH. Embryos can be stored at 20C in MeOH indefinitely. 4. Pre-Hybridisation Treatment Rehydrate in 50% MeOH / PTW (PTW = 1xPBS, 0.1% Tween) Post-fix for 15-20min in 3.7% formaldehyde/PTW, shaking gently. Wash 5 X 5 min with PTW. N.B. It is important to wash all 5 times and for 5 min to remove any traces of fix. Wash 10 min in 50% PTW : 50% hybe buffer Wash 10 min in hybe buffer Prehybridise at least 1 hour in hybe buffer at 70C (on a heat block or in an oven) 5. Hybridisation Add Dig-RNA probe in hybe (Usually diluted 1:1000, but test dilution for optimal results) to embryos using at least 100ul of probe making sure embryos are well covered and mixed with the probe. Leave overnight at 70C (on a heat block or in an oven) For all subsequent steps except the staining reaction, the embryos are gently rocked in an eppendorf in a nutator. 6. Washes: In morning remove and save probe (it can be used several times but does get diluted each time). Wash as follows (at 70C, warm up solutions first; no need to agitate): 1 X 30 min hybe 1 X 30 min 50% hybe / 50% PTW 4 X 30 min PTW 7. Antibody: Incubate embryos in 1:2000 anti-Dig Fab alkaline phosphatase coupled antibody (Boehringer Mannheim) in PTW. Rock the tubes on a rolling incubator. For lowest possible background when using very weak probes can preabsorb antibody (1:5 dilution for 1-4 hours at room temperature). 8. Washes: Wash 3 X 20 min with PTW Rinse 2 min with reaction solution: 100 mM Tris pH 9.5 100 mM NaCl 50 mM MgCl2 (N.B. Tris pH 9.5 must store as frozen aliquotes as Tris does not buffer pH9.5 very well and the solution will become pH <9.0 with time at room temp). 9. Colour reaction: Add 4.5ul NBT and 3.5ul X phosphate per ml of reaction solution (or premix supplied with Boehringer Mannheim kit). Transfer embryos to microtiter plates and incubate for 2 min to overnight (if o/n then in the dark). Use red stain from Vectastain for fluorescent substrate (but this diffuses a little). 10. Stop reaction by washing several times with PTW. Can then stain with other antibodies (e.g. anti-tubulin monoclonal, or Wheat Germ Agglutinin) Mount embryos in 80% Glycerol and seal slide with clear nail varnish. Hybe solution: 50% deionized formamide 5X SSC 100 ug/ml E. coli tRNA 50 ug/ml heparin 0.1% Tween 20 pH to 4.5 with Citric Acid NBT= 4-nitro blue tetrazolium chloride X-phophate= 5-bromo-4-choro-3-indolyl-phosphate Making Probe: Transcribe RNA probe using B.M. Dig labeling kit. The template can be from miniprep DNA (E.coli RNA is o.k.) and must be linearised with an appropreate restriction enzyme (for sense or anti-sense probes) and enzyme heat killed. Use about 1 mg of digest (no more than 2.5 ml) in 20ml probe reaction) Transcription reaction mix: linearised template (0.5mg/ml) 2ml NTP labelling mix (tube 7) 2ml 10x buffer (tube 8) 2ml RNase inhibitor (tube 10) 0.5ml ddH2O 11.5ml RNA polymerase (20U/ml) 2ml Incubate at 37 for 2h. Check yield on gel: load 0.5-1ml before and after incubation. Should see strongish novel RNA band as reaction should yield about 10mg DIG labelled RNA. Add 2ml DNAase (tube 9; 10U/ml). Incubate 15min/37C. Optional probe precipitation: Add 28ml dH2O. Ppt with 2ml 10M LiCl, 150ml ethanol at 20C Spin (13K 3 min); wash pellet with 70% EtOH, dry, and resuspend in 50ml H2O.