Aprendizaje en la Competencia de Comprensión de

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