«Joshua Frederick Coulcher UCL Submitted for the Degree of Doctor of Philosophy September 2011 Declaration I, Joshua Frederick Coulcher, confirm that ...»
The Digoxygenin (DIG) labelled RNA probe was prepared by adding 0.1µl-5µl to 100µl of hybridization solution. Probe concentration was optimized empirically, but typically optimum probe concentration was 0.5-1µl probe to 100µl Hybridization solution. The probe was denatured by incubating at 80 oC for ten minutes and then equilibrated at 60 oC in the water bath. The embryos were incubated in the probe solution for 42 hours at 60 oC.
After incubation with the RNA probe, embryos were washed in pre-heated hybridization solution several times over a period of two and a half hours at 60 oC.
Embryos were then washed with PBS-T several times over one hour at room temperature. Prior to antibody incubation, embryos were washed in blocking solution for 30 minutes. Alkaline phosphatase conjugated anti-DIG antibody (Roche, 11 093 274
910) was spun down at 13,000g for 10 minutes at 4 oC prior to use. Embryos were incubated with antibody diluted 1 (0.75U/µl):2000 (blocking solution). Antibody incubation was performed on a rocking platform overnight at 4 oC.
After antibody incubation, embryos were washed at least ten times over a period of 3 hours with PBS-T. Prior to staining embryos with NBT/BCIP, embryos were washed twice with staining buffer. Embryos were stained in the dark in 2% (v/v) NBT/BCIP solution in staining buffer. The staining reaction was stopped by several washes over a period of one hour. Embryos were stored at 4oC overnight prior to secondary staining for double in situ hybridizations (see below) or for up to 2 months prior to visualization by microscopy.
Double in situ hybridization of Tribolium embryos protocol Double in situ hybridization protocol used was the same as the single in situ protocol except two probes were added to the hybridization solution, one DIG labelled probe and one Fluorescein (FITC) labelled probe, and the protocol continues after the end of the first staining.
The alkaline phosphatase conjugated anti-DIG antibody was denatured by heating in 1ml of inactivation buffer for 15 minutes at 65 oC and left to cool for 20 minutes. Embryos were washed in 50% (v/v) inactivation buffer in PBS-T and then washed several times over a period of 30 minutes with PBS-T. Prior to incubation with alkaline phosphatase conjugated anti-FITC antibody (Roche, 11 428 338 910), embryos were incubated in blocking solution for half an hour. Alkaline phosphatase conjugated anti-FITC antibody was spun down at 13,000g for 10 minutes at 4 oC prior to use.
Embryos were incubated with antibody diluted 1:2000 in blocking solution. Antibody incubation was performed on a rocking platform either overnight at 4 oC or at room temperature for one hour. After antibody incubation, embryos were washed several times over a period of 3 hours with PBS-T. Prior to staining embryos with NBT/BCIP, embryos were washed twice with FastRed staining buffer. Embryos were stained in the dark in FastRed (Sigma). The staining reaction was stopped by several washes over a period of one hour. Embryos were stored at 4oC for up to 2 months prior to visualization by microscopy.
Tribolium fluorescent whole mount in situ hybridization protocol The fluorescent in situ protocol used was very similar to the single in situ hybridization protocol with a few modifications. The protocol used was a fluorescent in situ hybridization protocol (Kosman et al., 2004). Prior to post-fixation, embryos were treated with xylenes. Embryos were transferred from methanol to ethanol and washed three times in ethanol. Embryos were incubated in xylenes:ethanol (9:1) for one hour at room temperature. Post-fixation steps were increased to 25 minutes. Primary and secondary antibody incubation steps were performed. The washing steps for both antibody steps were the same as for the anti-DIG alkaline phosphatase conjugated antibody step. Numerous hapten, primary and secondary antibody combinations were tried to optimize the signal strength and reduce background (Alexa 405, 488, 555).
However, the only data presented in this thesis from fluorescent in situ hybridization was using a DIG labelled RNA probe with sheep anti-DIG antibody (Roche (1 333 089) and Alexa 555 Donkey anti-Sheep (Molecular Probes, A-21436).
Tribolium fluorescent whole mount in situ hybridization with Tyramide signal amplification.
The tyramide signal amplification (TSA) protocol is very similar to the single in situ hybridization protocol with a few modifications. Many of the reagents were provided with the TSA kit #12 HRP-goat anti-rabbit IgG Alexa fluor 488 tyramide (Invitrogen, T20922).
Achaearanea tepidariorum whole mount in situ hybridization.
Embryo collection and fixation Female spiders were mated with males. Cocoons that the female produced were separated. Embryos develop into spiders over the course of one week. Different stages of embryos were obtained by fixing at different times post cocoon production.
Embryos were removed from the cocoon and devitenilized by dropping 50% bleach with a pipette for 3 minutes and then washing thoroughly with water. Embryos were fixed in PEMS buffer with 6% formaldehyde for more than 5 hours, often overnight, washed several times with gradually increasing concentrations of methanol (mixed with PEMS). Several washes were performed with 100% methanol and stored at -20oC.
Embryos were devitellinized individually using sharpened tungsten wire (by holding over a flame and pulling apart), Dumont no.5 tweezers and Gilson pipettes.
Achaearanea in situ hybridization protocol The Achaearanea in situ hybridization protocol is nearly identical to the Tribolium in situ hybridization protocol. Some parameters were altered to try to optimize the in situ hybridization experiment: The concentration of probe was tested over a thousand-fold range (0.01µl-10 l labelled RNA probe/100µl hybridization solution). The incubation time of hybridization of the probe to the embryos was varied from overnight to one week. The incubation time of the anti-hapten antibody was varied from overnight to one week. The hybridization temperature was tested over a range of 55oC to 65oC. The concentration of formamide present in the hybridization solution was also increased to 75% (v/v) to reduce the stringency of RNA binding. The number and duration of washes post-hybridization and post-antibody incubation was substantially increased from several washes over two hours to several washes over more than six hours.
Achaearanea Tyramide signal amplification protocol Achaearanea in situ hybridization protocol is very similar to the Tribolium in situ hybridization protocol. Reagents were provided with the Renaissance TSA plus DNP AP system kit (PerkinElmer, NEL746A(AP)).
8.4 Microscopy Bright field Microscopy.
Prior to taking pictures, embryonic germ bands of Tribolium were dissected from yolk using a tool constructed with an eyelash hair attached with candlewax to a Gilson P200 pipette tip. Gene expression revealed by enzymatic in situ hybridization of Tribolium and Achaearanea embryos and cuticle preparations of Tribolium larvae was visualized with an Imager M1 microscope (Carl Zeiss). The majority of images were visualized using differential interference contrast (DIC) microscopy using magnification of x50, x100 and x200. Images were taken with Axiocam HRC (Carl Zeiss) and processed using Axiovision product suite Software (release 4.8.2). Images were edited with GIMP 2.6.10. GNU Image Manipulation Program (Copyright© 1995-2008 Spencer Kimball, Peter Mattis and the GIMP Development Team).
Confocal Microscopy Gene expression revealed by fluorescent in situ hybridization was visualized by confocal microscopy using an upright Leica TCS SPE confocal microscope. Laser excitation frequencies used were 405nm, 488nm and 532nm. Images were obtained and edited using Leica application suite (LAS-AF).
Scanning Electron Microscopy Tribolium embryos were fixed in 9.25% formaldehyde and devitinellized as described for the wholemount in situ hybridization protocol. Fixed embryos were rinsed in ethanol and immersed in HMDS and air dried overnight in an embryo dish.
Embryos were placed on 0.5” Aluminium pin stubs (Agar scientific). Embryos were sputter coated with gold in an EMSCOPE SC500. Scanning electron micrographs were taken in a JEOL JSM-5410LV scanning microscope at a magnification of 100 to 350 fold.
Images were acquired with DigitalMicrograph (Gatan).
Cuticle preparation of Tribolium larvae First instar larvae were collected by placing them on top of 300µm mesh and trapping those that crawl through into 100% glycerol. The larvae were collected and mounted onto a slide with a coverslip with 50% Hoyer’s medium and 50% lactic acid and incubated at 70oC for one week. Alternatively, larvae were placed in 90% lactic acid and 10% ethanol and incubated at 70 oC for at least one week, and then mounted on a microscope slide to be visualized with DIC microscopy or confocal fluorescent microscopy (larval cuticle auto-fluoresces at all visible wavelengths).
8.5 Parental RNAi in Tribolium
Female Tribolium pupae were injected with 1-5µg/µl dsRNA. dsRNA was injected with 1mm x 0.78mm borosilicate glass capillaries (CEI Harvard apparatus, GC100TF-10, 30-0038). Needles were pulled by a needle puller (Sutter instrument PNeedles were attached to an injection holder (Narishige, HI-7) and attached to a M1 micromanipulator (Helmut Saur). Injected female pupae were added to wholemeal flour plus yeast (5%) with male pupae for 1 week at 32 oC. Beetles were then transferred to white flour with 5% brewer’s yeast, pre-sieved with a 300µm sieve. Eggs were collected daily for three days and then the beetles were transferred back to wholemeal flour for three days. Embryos were allowed to develop until the 1st instar larval stage and were collected for cuticle preparations and epistatic interaction assayed by wholemount in situ hybridizations with appropriate genes. This cycle of medium transfer was repeated until no observable phenotypes were present.
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