-To the apple juice plate add a solution of 3% chlorox for 1 min., while loosening the embryos with a paint brush.
-Pour the embryos into a filter basket and wash with water.
-Blot dry on a kleenex.
-Take the mesh and immerse in 2.0ml methanol and 2.0 ml heptane in a 15 ml centrifuge tube.
-Get as many larva off the filter as you can.
-Shake hard for 30sec, the larva should sink to the bottom.
-Remove the heptane and methanol.
-Wash 2X with 1ml of methanol.
-Take larva up using a P200 with a cut off pipette tip, and put on a cleaned microscope slide (washed with ethanol and dried with a kleenex).
- Blot the excess methanol with a kleenex, and let the residual evaporate.
-As soon as the methanol evaporates add 80ul of 1:1 Hoyer's mountant: lactic acid.
-Incubate at 60°C overnight.
Hoyer's mountant
- 30g acacia (gum Arabic)
- 200g chloral hydrate
- 50g water
- 20g glycerol
-Change apple juice plate and collect embryos for 2/3 hr.
-Change plates and take the plate and incubate at 23°C for 3 hr.
-Add room temp. water to the plate and with a paint brush suspend the embryos.
-Collect embryos on a mesh filter.
-Wash with room temp. water, trying to corral the embryos to one side of the mesh.
-Blot the mesh dry on a kleenex.
-Roll up the filter and place in a 1.5 ml eppendorf tube.
-Close the cap and submerge embryos in a 36.5°C water bath for 18 min. when the embryos are 3 to 3 2/3 hrs old. Such that the average time of heat-shock is 3 1/3 hr.
-Remove the filter from the tube and place on a apple juice plate.
-Let develop for 24 h at 23°C.
-add 3% chlorox: many larvae will have hatched and left the mesh, so make sure all the hatched and unhatched larvae are removed from the mesh.
-Strain through the mesh
-Blot dry.
-Prepare the cuticles as before starting at immersing the larvae in the heptane;methanol solution.
-Collect and dechorionate embryos as usual.
-Fix by dipping the collection screen in a scintillation vial containing 5ml heptane, 0.5ml formaldehyde (carcinogen), 4.5ml PBS.
-Incubate at room temp. for 20 min. swirling occasionally.
-Remove the embryos from the interphase with a P200 with a cut off plastic tip and transfer them to an eppendorf tube, be sure no aqueous phase is transferred.
-Wash twice with heptane.
-Add 0.5ml of heptane and then 0.5ml of methanol.
-Vigorously shake for 1 min., the embryos should sink to the bottom.
-Remove heptane and interphase with a micropipette.
-Using the micropipette wash embryos twice with methanol.
-Split embryos into two tubes per strain/genotype (4 tubes total).
-Store embryos in methanol at -20°C.
-Wash the embryos stored in methanol 2-3X with Blotto.
-Block for 20min in Blotto, i.e. incubate for 20min in Blotto on the tube rocker.
-Remove Blotto.
-Add 400 ul anti monoclonal antibody (Ab) solution (Ab in Blotto), and incubate at room temperature on the tube rocker for 2 hr.
-Remove the Ab solution and wash briefly 3x with Blotto.
-Transfer the embryos to a new tube using a P200 with a cut off tip, and using a micropipette wash 3x with Blotto for 20min each on the rocker.
-Remove Blotto
-Add 400ul secondary Ab (vectastain elite) in Blotto for 1 hr.
-Wash 3X with PBT briefly.
-Transfer embryos to a new tube using a P200 with a cut off tip.
-Wash 3X for 15min with PBT on the tube rocker.
-Add 500 ul ABC (vectastain elite) reaction for 30 min. on the tube rocker.
-Wash 3X briefly with PBT
-Transfer the embryos to a new tube using a P200 with a cut off tip.
-Wash 2X for 15min with PBT on the tube rocker.
-rinse with 1ml of peroxidase buffer.
-To 1ml of peroxidase buffer add 10 ul of DAB reagent (DAB is extremely carcinogenic, wear gloves lab coat and safety glasses, put all contaminated solutions, tips and so forth in the bleach solution provided). Close lid of tube and invert to mix the solution.
-Watch color develop in a weigh boat under the microscope; when dark enough remove staining solution and wash 3X with PBT.
-Remove PBT and add 80ul mountant.
-Mount embryos on a slide, and seal with nail varnish.
PBS
PBT 1XPBS
- 0.1% Tween 20
- Blotto 1XPBS
- 0.1 % Tween 20
- 0.5% skim milk powder
- 0.02% NaN3 (poisonous)
Mountant
- 80% glycerol
- 20mM Tris HCl pH 8.0
-Wash embryos with PBT (.1% tween 20) 1X5Min ;1X20min.
-Rinse with water
-Add ice cold acetone 200 ul for 5 min (no longer than 10 min) keep tube on ice.
-Wash with PBT 2X10min
-Rinse twice with 200ul of 50% PBT/50% hybridization solution.
-Rinse with 100µl of 100% hybridization solution.
-Prehybridize for 1hr at 55°C in 100ul of hybridization solution.
-Remove the prehybridization solution and add 50ul of hybridization solution+5ul wg RNA probe.
-Hybridize overnight at 55°C.
-Remove the hybridization solution+wg probe, and wash the embryos with hybridization solution for 20 min. at 55°C twice.
-Remove the solution and wash with hybridization solution/PBT (1:1) for 20 min. at 55°C.
-Wash 2X with PBT for 20 min. each at 55°C (change tubes after second wash).
-Incubate with anti-DIG antibody (a 1:2000 dilution in PBT) for 2 hr. at room temperature on the tube rocker.
-Rinse twice and wash twice for 20min. with PBT (change tubes after rinses).
-Rinse 2X with AP buffer.
-Add 9ul of NBT and 7ul of X-phosphate (both these chemicals and the buffer are to be handled with care) to 1ml of AP buffer.
-Let the color reaction develop 1-4 hr at 25°C.
-Stop the reaction with three PBT washes.
-Do the following dehydration/rehydration series 2min each 50% ethanol, 70%ethanol, 90%ethanol, 100%ethanol, 90%ethanol, 70%ethanol, 50%ethanol, 25%ethanol.
-Add 80ul mountant.
-Mount embryos on a slide, and seal with nail varnish.
fix solution
- PBT
- 5% formaldehyde (carcinogenic)
Hybridization solution 50% deionized formamide
- 0.1% Tween20
- 100ug/ml total yeast tRNA
- 100ug/ml heperin
- 150mM NaCl
AP buffer
- 100mM NaCl
- 50mM MgCl2
- 100mM Tris pH9.5
- 0.1% Tween 20
-Collect embryos from yeasted apple juice plates
-Dechorionate in 3% chlorox for 1-2 min. swirling occasionally
-Wash embryos thoroughly with water.
-Blot the mesh dry on a kleenex.
-Fix by dipping the collection screen in a scintillation vial containing 5ml heptane, 0.5ml formaldehyde (carcinogen), 4.5ml PBS.
-Fix for 15-20 min. at room temp. by swirling.
-Meanwhile preincubate 0.5ml aliquots of staining solution at 37°C. Add 10 ul 10% X-Gal (5-bromo-4-chloro-3-indolyl -D-galactoside) in DMSO and incubate at least 10 min. Just before use, centrifuge for 3min in an eppendorf microfuge to pellet crystals.
-Remove the embryos from the fix solution and place in an eppendorf tube.
-Wash 3X with PBS+tween, when the heptane is washed away, the embryos no longer aggregate or stick to the side of the tube.
-Resuspend embryos in staining solution (precentrifuged) and incubate for overnight .
-Remove the staining solution and wash 2-3 times in PBS+tween to remove most of the crystals.
-Resuspend the embryos in mountant and mount.
PBS+tween
PBS+0.3% tween 20
Staining solution
- 10.0mM Na phosphate pH 7.2
- 150.0mM NaCl
- 1.0 mM MgCl2
- 3.1 mM K4(FeII(CN)6)
- 3.1 mM K3(FeIII(CN)6)
- 0.3% tween 20