ZONA LIMPIA

Wholemount in situ hybridisation was carried out essentially as described by Wilkinson (1992).

2.18.1 Synthesis of riboprobes

Riboprobes were synthesised from pBluescript or pGEM-T clones of the fragments of interest. Clones were linearised by digestion with an appropriate restriction enzyme, such that the RNA polymerase would cease RNA synthesis immediately after

transcribing through the insert. Following purification, 0.5-1 pg of linearised plasmid DNA was transcribed in a 20 pi reaction containing Ix transcription buffer, 10 mM DTT, 2 pi digoxigenin-labelling mix (Boehringer Mannheim), 50 Units placental ribonuclease inhibitor, and 10 Units of either T3, Sp6 or T7 RNA polymerase. T7 and T3 polymerases transcribe from pBluescript clones, in opposite directions, therefore generating sense or antisense probes, depending on the orientation of the insert. Similarly, Sp6 and T7 RNA polymerases were used to generate sense and antisense probes from pGEM-T clones. The transcription reaction was incubated at 37°C for 2.5- 3 hours, then 1 pi was electrophoresed on a 1 % agarose gel to check that the reaction had worked successfully, with the RNA band appearing 10-20 fold more intense than the plasmid band. If the reaction was successful, the RNA was precipitated by the addition of 100 pi TE, 10 pi 4M LiCl and 300 pi ethanol, with incubation at -20°C for at least 30 minutes. The RNA was pelleted by centrifugation at 13,000 rpm for 10

minutes, washed in 70% ethanol and allowed to air dry. The probe was redissolved in TE at approximately 0.1 pg/pl and stored at -20°C.

2.18.2 Embryo pretreatments and bybridisation

The embryos were dissected from the yolk sac and amnion and fixed at 4°C overnight with 4% paraformaldehyde (PEA) in PBS. Embryos were dehydrated through a series of methanol solutions (25, 50, 75, 100% all in PBT (PBS with 0.1% Tween-20)) at 4°C, to prevent the formation of air bubbles during the later hydrogen peroxide treatment, which would damage the embryos. If not to be used immediately, the embryos were stored in

100% methanol at -20°C. Embryos were then rehydrated through the methanol series and washed twice with PBT. Endogenous peroxidase was blocked by washing in 6% hydrogen peroxide in PBT and embryos were treated with 5 pg/ml proteinase K to

increase the penetration of the probe. The length of time of this digestion was

dependent on the age of the embryos; E8.5 embryos were treated for 1 minute, E9.5 for 2 minutes, and E10.5 for 6 minutes. These times were found to be just sufficient to allow the probe to penetrate without causing reopening of the neural tube. The proteinase K digestion was inhibited by transferring embryos into 2 mg/ml glycine in PBT, and embryos were post-fixed with 4% PFA/0.2% glutaraldehyde in PBS. The embryos were prehybridised for at least 1 hour in prehybridisation solution (50% formamide, 5 x SSC pH 4.5, 50 pg/ml heparin, 50 pg/ml yeast transfer RNA, 1% SDS) at 70°C. If necessary, embryos were stored in prehybridisation solution at -20°C until required; in this case, the incubation at 70°C was performed immediately prior to the hybridisation reaction. Embryos were hybridised overnight at 70°C in 1 ml fresh prehybridisation solution containing the digoxigenin-labeled RNA probe at 1 pg/ml.

2.18.3 Post-hybridisation washes

Following hybridisation, the embryos were transferred to a 15 ml Falcon tube to enable all the washing steps to be performed in a volume of 10-12 ml. The hybridisation solution was stored at -20°C and reused two or three times. Embryos were washed twice in solution 1 (50% formamide, 5 x SSC pH 4.5, 1% SDS), then twice in solution 2 (50% formamide, 2 x SSC pH 4.5), for 1 hour each at 70°C. They were then washed in TBST (137 mM NaCl, 2.7 mM KCl, 25 mM Tris HCl pH 7.5, 1% Tween-20, 0.48 mg/ml Levamisole (Sigma)) three times for 5 minutes each at room temperature, then blocked with 10% sheep serum in TBST for 90 minutes at 4°C to prevent non-specific binding of the antibody. The alkaline phosphatase conjugated anti-digoxigenin antibody (Boehringer Mannheim) was diluted 1 in 1000 with TBST and 0.1% sheep serum then preabsorbed by incubating with embryo powder (homogenised E l 2.5 mouse embryos, washed with acetone), for 1 hour at 4°C. The antibody was then removed from the embryo powder by centrifugation, diluted with an equal volume TBST containing 1 % sheep serum, then filtered through a 0.4 pm Millipore filter. Embryos were incubated overnight at 4°C with 2 ml preabsorbed antibody solution, then washed at room

temperature for 24 hours with 9 or 10 changes of TBST. The TBST was replaced with NTMT (100 mM NaCl, 100 mM Tris HCl pH 9.5, 50 mM MgCl2, 0.1% Tween-20), and

the colour reaction produced by incubation in the dark with 4.5 pi 4-nitro blue

1 ml NTMT. After completion of the colour reaction the embryos were washed in PBT and photographed using a Zeiss SVl 1 stereomicroscope before processing for

sectioning.

2.18.4 Vibratome sectioning of wholemount embryos

Embryos were equilibrated overnight in gelatine-albumin mix (0.45g gelatine 300 Bloom, 27g chicken egg albumin grade II and 18g sucrose in 80 ml PBS) then orientated and set in a plastic mould with the addition of glutaraldehyde to 2.5%. The mix was allowed to set for at least 1 hour, then 50 pm thick sections were cut using a Series 1000 vibratome (Agar Scientific). Embedded embryos were sometimes stored in PBS at 4°C for several months before sectioning. Sections were mounted onto slides with 50% glycerol/PBT and examined using differential interference contrast (DIG; Nomarski) optics on an Axiophot microscope (Zeiss).

CHAPTER 3