Marek Mlodzik

FIXATION AND PRETREATMENT FOR IMAGINAL DISC WHOLE MOUNT NONRADIOACTIVE IN SITU HYBRIDIZATION

ºimaginal disc; non-radioactive in situ hybridization;
The following protocol is based on the method described for whole mount embryo hybridization (see above).
1) Dissect discs from late 3rd instar larvae in PBS.
2) Fix dissected material for 15'-20' in 4% paraformaldehyde, PBS, 0,5% Triton X-100 on ice in Eppendorf-tube, while you continue to dissect more discs.
3) Fix for additioinal 20' at RT (same fixative as above) after you have finished dissecting.
4) Following fixation, wash the discs 3 times for 5' in PTw at RT.
5) ProteinaseK digestion:
12.5 µg/ml enzyme in PTw (1/4 of the concentration that is used for embryos)
usually between 4' and 10' at RT is fine, depends on batch of proteinaseK
when new batch: titrate time !
The longest digestion that still retains reasonable morphology gives the best results.
6) Wash digested discs in PTw, 2mg/ml glycine: 2 times 5' at RT, followed by two 5' washes in PTw.
7) Postfixation: 20' at RT in 4% paraformaldehyde
0.2% glutaraldehyde
PTw
8) Wash fixed discs in PTw 4 times 5' at RT.

Prehybridization:
1) PTw : hybridization buffer (1:1) for 10' at RT
2) Hybridization buffer, 10' at RT
3) Hybridization buffer, 1 hour at 45 C
4) Hybridize o/n at 45 C, in Eppendorf tube without shaking

Buffers:

PBS (10X stock) 76.1 g NaCl
18.8 g Na2HPO4.7H2O (9.9 g if anhydrous)
4.1 g NaH2PO4.H2O

H2O to 1 litre

PTw PBS containing 0.1% Tween

Hybridization Buffer same as for embryos (see above)

Probe and Detection:
Probe can be made either by random-oligo priming or alternatively by PCR generating single stranded labelled DNA (see above).
More recently, a digoxigenin;-labelling kit for RNA probes is also available.

Detection is done as described for embryos (see above), all steps in Eppendorf tubes.

Comments:
The success of the method depends largely on the abundance of the respective transcripts. In our experience, detection of transcripts that are found at 1:50,000 (or more abundant) in the eye disc cDNA library usually work fine, e.g. glass (1:5000) or scabrous (1:20,000). For transcripts with lower abundance, e.g. seven-up (1:100,000) or sevenless (1:100,000), this method works in about 1 out of 2 experiments.

GOOD LUCK !