This protocol is based on the one described by Row eer al (1992).
2.2.6.1 Tissue Preparation
Chick em bryos were dissected and rinsed in icy-cold PBS, pinned flat out on
w ax dishes and fixed overnight in 4% PFA in PBS at 4 °C . For embryos older than
stage 20, heads were cut o ff and processed further; w hole embryos were used if
younger. Individual eyes of em bryos older than stage 30 were dissected out and
processed.
The tissue w as put through two 30min changes o f PBS at 4 °C , followed by a
15min. w ash in 1:1 ethanol/PBS at the same temperature. They were then washed twice
in 70% ethanol at room temperature for 15min each. The final dehydration series
involved 30m in. incubations at room temperature in 85% ethanol, 95% ethanol,
absolute ethanol (twice) and toluene (twice). The tissue w as then incubated in molten
w ax for 20 m in. at 60°C . This w as repeated twice before embedding the tissue, in the
right orientation, in fresh m olten wax and allowing it to cool to room temperature. The
blocks w ere stored at 4^C in a sealed box with desiccant for up to 4 months.
2 .2 .6 .2 Preparation of slides and sections
Glass slides were w ashed in Teepol detergent and rinsed thoroughly, first in tap
w ater and lastly in double deionised water. The slides w ere then incubated in 10% HCl
for 20m in and put through 3 rinses of double deionised water. They were then baked at
180 °C for about 2 hours and allowed to cool to room temperature before coating with
TES PA (3-am inopropyl triethoxysilane). To accomplish this, the slides were dipped in
a solution o f 2% TESPA in acetone, acetone, acetone and double deionised w ater for
The slides were placed in a 42^C oven for about an hour. The prepared slides
w ere w rapped in aluminium foil and used within four days.
For sectioning, 8pm sections of tissue were cut on a microtome and floated on
to the coated slides at 45 °C . The sections were allowed to dry down at 45 °C for 2
hours and baked onto the slides at 60°C overnight. Sections were stored at 4^C in slide
boxes containing desiccant,
2.2.6.3 ^^S-labelled Probe preparation
The probes were synthesised according to Devlin et al, 1988.
Chicken BM P-4 cDNA w as linearised with B am W l (antisense) or HindiW
(sense). G H 6 cDNA w as linearised with HindiW (antisense) or E coR l (sense), while
linearisation of Shh was performed with 5'fl//(antisense) o r N o tl (sense). The linearised
plasm ids w ere subsequently treated with phenol/chloroform , ethanol precipitated and
w ashed in the same way. The D N A w as finally resuspended in sterile w ater containing
lOmM dithiothreitol (DTT) at « Ip g /p l.
25 pi transcription reactions were set up containing 5 pi of 5x reaction
buffer, 0.5pl of IM D TP, 1.2pl of lOmM GTP, 1.2pl o f lOmM ATP, 1.2pl of lOmM
U TP, I p l o f 50pM GTP, 5 p g of linearised plasmid D N A and made up to 25pi w ith
sterile w ater. Finally, 7pl of S GTP was added, together with 0.5pl of RNAsin and
1.5pi o f the relevant RNA polym erase (Tg for the B m p -4 and GH6 antisense; Sp 6 for
Shh antisense; Ty for the B m p -4 , G H 6 and Shh sense). The reaction mixture w as left
for one hour at room temperature.
0 .5pl of RN Asin, I p l lOmg/ml tRNA, 0.5pi IM DTT and 0 .5 pi stock BRL
RN ase-free DNase I w as mixed in and the reaction mixture incubated at 37°G for
lOmin. After this, 95pi of sterile w ater and lOpl of 5M LiGl was added, the mixture
The RN A w as pelleted by centrifugation in a m icrofuge, w ashed w ith lOmM
D T T in 70% ethanol and left to dry. Finally, it was resuspended in 50pl of 50mM DTT
in sterile water and 0.5pi was counted in a scintillation counter with "Ecoscint".
7 8
The average number of counts obtained w as betw een 3x10 and 1x10 cpm
total.
Because the length of the RNA produced by transcription w ould have reduced
penetration of the tissue sections, it was necessary to subject some o f the probes to
alkaline hydrolysis. All the probes but the G H 6 probe required hydrolysis. 50,ul o f
hydrolysis buffer (SOmM sodium hydrogen carbonate, 120mM sodium carbonate pH
10.2 and lOmM DTT) was added to 50pl of RNA probe and the mixture left at 60°C
for the requisite length of time (20min for the BM P probes, 27min for sh h ). The
reaction was halted with 50pl of neutralising buffer (0.2M sodium acetate, 1% glacial
acetic acid and lOmM DTT) and precipitated with 15pl 3M sodium acetate (pH 5.2) and
300,ul cold absolute ethanol at -70°C for lOmin. After centrifugation, the probe w as
w ashed with lOmM DTT in 70% ethanol, dried and resuspended in 50pl sterile water
w ith lOmM DTT.
The probe w as stored at -70^C and counted again before use.
2 .2 .6 .4 Pretreatment
The procedure was performed as described by Row e et al (1991).
The slides were allowed to warm to room temperature from 4 °C while still in
their sealed box. They were dewaxed in fresh xylene for 15min and then rehydrated for
2 m in each in absolute, 95%, 90%, 80% , 60% and 30% ethanol and double deionised
w ater.
The following steps were carried out in glass dishes previously baked at 180°C
for a minimum o f two hours. All buffers were autoclaved before use and effort w as
m ade to keep everything RNAse-free. Where temperature is not mentioned, steps w ere
The slides were washed twice for 2 m in in sterile w ater and left in 1/49 H Cl for
about 15 min They were then rinsed for 5 min in 2xSSC and treated with 5pig/ml
proteinase K in lOOmM Tris pH 7.5, 50mM EDTA, 3 7 °C before being incubated in
2mg/ml glycine in PBS for 2 min at room temperature. This was followed by another
two 2min w ashes in PBS and 20min fixation in 4% P FA in PBS. Tw o m ore 2min
w ashes in PBS w ere followed by a lOmin acétylation step in a solution o f O.IM
triethanolamine, 1/400 acetic anhydride that had been mixed immediately before use.
There was then another 5nun incubation in PBS, a 2min w ash in sterile w ater and
finally dehydration through 30%, 60%, 80%, 95% and absolute ethanol (2 min each).
The slides were air-dried under aluminium foil for one hour.
2.2.6.5 Hybridisation
A hybridisation mixture consisting of Ix D enhardt's solution, 10% (v/v)
deionised form am ide, 50mM DTT, 50mg/ml yeast total RNA (phenol/choloroform
extracted stock), 50pg poly A RNA and probe diluted to about 1x10^ cpm per pi w as
prepared. The mixture was vortexed, spun down, vortexed and spun dow n again
before use to rem ove all air bubbles. It was heated to SO^C for 3 min. and cooled on ice
before use.
The required amount of hybridisation mixture (50pl for small sections; 70pl for
large) w as spotted directly on to the dry slides w ith a Gilson m icropipette, and a
coverslip lowered slowly over the slide so that the m ixture w as spread to all sections on
the slide.
The slides were rested on pasteur pipettes inside sealable plastic boxes. 2xS S C ,
50% formamide saturated 3mm paper was placed at the bottom of the boxes. Dry 3mm
paper w as taped to the lids and the boxes sealed w ith tape and kept in a 5 5 °C oven