1. It is essential to avoid the degradation of the target of ISH, cellular RNA, prior to hybridization. Ribonuclease contamination should be avoided by autoclaving solutions; baking glassware and consumable supplies and reagents should be kept exclusively for ISH. Disposable gloves should be worn.
2. Extraneous transcripts have been reported to occur during the preparation of the riboprobe when the template contains 3' protruding ends, therefore do not use restriction enzymes that generate 3' overhangs. If there is no alternative restriction site the 3' overhang should be converted to a blunt end using Klenow DNA polymerase.
3. The mixture for the in vitro transcription protocol should be kept at room temperature during the addition of each successive component, since DNA can precipitate in the presence of spermidine (present in the transcription buffer) if kept at 4°C.
4. Histolene used for the initial dewaxing of sections can be kept for use in mounting slides in DePex.
5. The hybridization temperature may need to be altered. Oligonucleotide probes are hybridised at 30-37°C overnight (2,6,7). cRNA probes are hybridized at40-70°C (4,7).
6. To pre-absorb antibody: Weigh out 45 mg chick embryo (or appropriate tissue) powder into a microfuge tube, add 7.5 mL TBST and heat at 70°C for 30 min. Cool on ice and add 75 |L sheep serum and 15 |L of anti-DIG Fab fragments. Shake at 4°C for 1 h to preabsorb antibody. Spin down solids from powder for 10 min. Remove supernatant and dilute it to 75 mL with 1% sheep serum in TBST. Antibody can be stored at 4°C with 0.02% sodium azide and reused at least once.
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