8.) MATERIALS & METHODS
go to: table of contents - preface - abstract- intoduction - chapter 2 - chapter 3 - chapter 4
chapter 5 - chapter 6 - conclusions - materials & methods - references
8.1 Flystocks and Staging
Oregon-R was used as a wild-type strain. Embryos were raised at 25ºC and staged
according to (CAMPOS-ORTEGA & HARTENSTEIN, 1997), if not stated otherwise. The
age of specimens was referred to in hours after egg laying (AEL) at 25ºC.
Precise staging was achieved by selecting embryos immediately after formation of
the second midgut constriction (“3-part-gut” at 13 hr AEL +/- 10 min at 25ºC)
and subsequent incubation for further time at 25ºC. Mutations were maintained
over standard balancers with GFP markers.
8.2 Dechorionation and Wholemount Fixation
Embryos were harvested from agar plates and attached to laying pots. The eggs were exposed to bleach from a stock solution for about one to three minutes and washed several times in a cage with tap water in order to remove the chorion. Then the embryos were transferred into a glass vial with 500 mL heptane, 400 mL PBS (0.075 mL, pH 7.2) and 100 mL formaldehyde (stock solution at 40%), covered and kept under rotation for approximately 30 minutes. Then the embryos were transferred again into a 2 mL Eppendorf vial with 600 mL methanol and 600 mL heptane, using a pipette. They were vortexed for one minute in order to remove the vitelline membranes and washed in methanol for 3 times, approximately 15 minutes each. These washing steps were followed by four to five more with PBS-Tx (0.3%, 0.075 M, pH 7.2).
Fixed and washed specimens were blocked in PBS-T/BSA (0.075 M phosphate buffered
saline at pH 7.2 with 0.3% triton-X and 5 % bovine calf serum) for 15 minutes.
Then they were incubated with the primary antibody in phosphate buffered saline
(0.075 M, pH 7.2) with 0.3% Triton-X overnight at 25ºC. After this, the antibody
solution was removed and the specimens were rinsed in blocking solution
(PBS-T/BSA) three to five times, 10 minutes each.
8.4 DAB Development
15 mL DAB were solved in 300 mL PBS (0.075 M, pH 7.2). The specimens in a watchglass were exposed to about half this volume for 5 minutes to allow the DAB to penetrate the tissue. In the meantime, 5 mL of 30% H202 were diluted in 100 mL PBS and 10 mL of this solution added to the remaining DAB. For black precipitate with enhanced contrast, 10 mL of aqueous 8% NiCl solution were mixed with the DAB solution additionally. The final solution was added to the watchglass and the progress of the development was checked under a stereo microscope and stopped with PBS. The tissues were then rinsed three times in PBS. Specimens were finally mounted using DPX or more often Araldite.
8.5 Dehydration and Mounting
Specimens that were previously treated (wholemount fixation or flat preparation and immunohistochemistry) were dehydrated in ethanol (30%, 50%, 70%, 90%, 100%, 100%, 100%; approximately 10 min each) and either cleared with histoclear (15 min) for DPX, or directly mounted in Araldite. Slides with Araldite were polymerised overnight at approximately 60 ºC (on a hotplate).
8.6 Flat Preparations
Embryos were collected, dechorionated and staged as described in (LANDGRAF et al., 1997). Under phosphate buffered saline (0.075 M phosphate buffer, pH 7.2) the embryos were transferred to polylysine-coated coverslips, lifted out of the vitelline membrane with a glass needle, cut open along the body-axis (typically along the dorsal midline) and then attached to the coverslip. Using a glass needle that was attached to a mouth-piece (a plastic pipette tip) and a rubber tube, the gut and fat body were removed by gentle suction and the embryos were flattened by blowing a stream of saline over them. This way, the body wall was opened like a book and the CNS was exposed. The trachea were lifted up and either “pinned” away from the specimen or removed; the same treatment was applied to the hindgut. Flat preparations were fixed in 3.7% formaldehyde for 3 (dye-fills) to 30 (antibody labelling) minutes and rinsed in PBS (dye fills) or PBT (antibody labelling).
8.7 Microscopy and Imaging
Specimens were analysed on a Zeiss Axiophot Photomicroscope using transmitted light and incident-light fluorescence. Images and movies were collected using the Zeiss AxioCam Mrm CCD camera and Zeiss Axiovision 4.0 imaging software. Confocal Microscopy was performed on a Leica SP1 using 63x oil or water immersion objectives. In some cases, overlays or z-stack-projections were generated and processed with ImageJ (United States National Institute of Health software, Bethesda, Maryland, US) or Adobe Photoshop software (Adobe Systems Incorporated, San Jose, California US).
8.8 Dye Fills and Injections
DiI backfills of NMJs: Lipid-soluble carbocyanoine dye
1,1’-dioctadecyl-3,3,3’,3’-tetramethyl indocarbocyanide perchlorate (DiI) (Molecular
Probes, Eugene, Oregon, US) was dissolved in vegetable oil as described by (BOSSING
& TECHNAU, 1994) and backfilled into sharpened glass capillaries, which were
then bevelled and mounted to a piece of rubber tubing and a plastic syringe.
With use of a 63x water immersion lens on a Zeiss fixed-stage microscope or an
Olympus BX-50-WI and a hydraulic micromanipulator (Narishige, Tokyo, Japan), a
small droplet of DiI was manually deposited on a particular neuromuscular
junction (NMJ) by applying pressure on the syringe. If necessary, motor neuron
axons and NMJs were highlighted with a GFP conjugated anti-HRP antibody and the
drop of dye was applied under UV light. Then the dye was left to diffuse
overnight at 4ºC. To access NMJs of external muscles, internal muscle layers
were removed surgically using a glass needle. Preparations were analysed using a
Zeiss Axiophot microscope with UV (see 8.7 Microscopy and Imaging).
8.9 Phalloidin Injections
1 mL of phalloidin (Alexa Fluor 568 phalloidin solution in 1.5 mL methanol) was mixed with 100 mL of a solution of 7.4% formaldehyde in phosphate buffered saline (0.15 M, pH 7.2) and 100 mL methanol. This resulted in a final solution of 1 mL phalloidin in 3.7% formaldehyde in PBS (0.075 M, pH 7.2) and 50% methanol (1:200). The methanol had proved to be crucial to permeabilise the muscle sheath. This solution was sucked into glass capillaries that were connected to a 10 mL syringe through a piece of rubber-tubing. The tip of this capillary was then pierced through the bodywall of third instar larvae on the dorsal midline approximately at segment A5 or A6 to get as close to the posterior end as possible without distorting the muscles in A8/9. A small amount of the phalloidin solution was injected, so that the larva expanded. The pressure was kept for 30 seconds, after which the capillary was retracted and most of the phalloidin ran out of the larva. This procedure was repeated two or three times and the larva was then examined under fluorescence (see Material and Methods 8.7).
8.10 Larval Preparations: Glue preparations and CNS Dissections
Larvae were anaesthetised by cooling them at -20ºC for about 5 minutes. Then
they were transferred to a Sylgard (Dow Corning)-coated coverslip and fixed at
both their anterior and posterior ends, using cyanoacrylate glue (Histoacryl;
Braun, Melsungen, Germany) under phosphate buffered saline (0.075 M, pH 7.2) (BAINES
& BATE, 1998). Larvae were then opened dorsally using sharp tungsten needles and
glued flat to the sylgard coverslip. Gut and fat body were removed by sucking
and blowing saline through a glass capillary attached to a short piece of rubber
tubing in order to expose the ventral nerve cord and the muscle field. The
tracheae were pulled off and glued to the sylgard. The specimen was then viewed
using a 63x water immersion lens combined with Nomarski optics (see also 8.7
Microscopy and Imaging).
8.11 Heatshocks and Generation of MN-clones
OK371-Gal4 (K.G. Moffat, J.B Connolly, J. Keane, S.T. Sweeney, and C.J. O'Kane,
unpublished; see also MAHR & ABERLE, 2005) flies were crossed with flies
carrying the heatshock-Flipase and UAS-FRT-STOP-FRT-CD8-GFP constructs
(heatshock flipase; UAS-Stop-CD8-GFP/Cyo; Tm6/MKRS) (WONG et al., 2002). Embryos
were heatshocked on an apple juice agar plate at 37ºC for 30 min to generate a
fractionated OK371 pattern with individual, identifiable motorneurons. The
heatshocked embryos were raised on the apple juice agar plates with yeast at
25ºC and examined as third instar larvae by simply anaesthetising them (cooled
at -20ºC for about 10 minutes) and transferring them into a drop of saline under
a coverslip (see also 8.7 Microscopy and Imaging).
return to "thesis overview"