Method prepared by Grace Panganiban:


RNA in situ protocol for DNA and RNA probes

This procedure was adapted from one originally described by Tautz and Pfeifle (1989). It makes use of digoxigenin labelled probes and alkaline phosphatase-conjugated anti-digoxigenin antibody fragments (Boehringer Mannheim Biochemicals) to detect the hybridizing RNA in whole embryos. The major modification made was to increase the number of post-hybridization washes in hybridization buffer (50% deionized formamide, 5x SSC, 200 ug/ml tRNA, 100 ug/ml sonicated, boiled salmon sperm DNA, 0.1% Tween-20) and the duration of the hybridization buffer washes. This dramatically reduces the background, because the hybridization buffer is approximately 20oC more stringent than the PBT used for the majority of the washes in the original protocol.

DNA probe conditions are given; RNA probe conditions are indicated with *s.

Hybridization buffer* PBT*
50% deionized formamide 10 mM KPO4
5x SSC 140 mM NaCl
200 ug/ml tRNA 0.1% Tween 20
100 ug/ml sheared salmon sperm DNA pH 7.0
0.1% Tween 20

*When using riboprobes, make up the hybridization buffer and PBT with RNAse-free solutions and substitute 1 mg/ml glycogen for the tRNA.

Fixative Staining buffer
1x PBS (from 10x stock) 100 mM NaCl
9.4% formaldehyde (from 37% stock) 50 mM MgCl2
50 mM EGTA 100 mM Tris 9.5
0.1% Tween 20
5% formaldehyde in PBT
(we use 6.8 mls of 37% formaldehyde + 43.2 mls of PBT/ 50 mls of posfix)

Other reagents needed:
Drosophila saline (0.7% NaCl; 0.03% Triton X-100)
50% bleach heptane
methanol absolute ethanol (EtOH)
xylene 5 mgs/ml proteinase K
PBT + 2 mgs/ml glycine PBT + 50 mM EDTA
nitroblue tetrazolium (NBT) (75 mgs/ml in DMF)
X-phosphate (50 mgs/ml in DMF)
10-90% glycerol, 50 mM EDTA (for clearing and mounting embryos)

1) Wash staged embryos in Drosophila saline. Rinse with deionized water (diw).

2) Dechorionate in 50% bleach for 2-3 min. Rinse thoroughly with diw.

3) Fix for 40 min. in 50% heptane: 50% fixative.

4) Replace the fixative with methanol and devitellinize the embryos by shaking. Devitellinized embryos will sink to the bottom of the tube.

5) Aspirate off the heptane and methanol. Rinse the embryos 2x with methanol and 4x with EtOH. The embryos can be used immediately or stored at -20oC in EtOH for at least one year.

6) Prior to staining, rinse embryos in 50% EtOH: fifty percent xylene and soaked in xylene for two hours.

7) Rinse embryos 1x with 50% EtOH: fifty percent xylene, then 3x with EtOH, 1x with methanol, 1x with 50% methanol: 50% postfix, and 1x with postfix.

8) Postfix the embryos for twenty min.

9) Wash the embryos 3x with PBT.

10) Incubate for 3-5 min. with 50 ug/ml proteinase K in PBT**.
**When using riboprobes, it is necessary to reduce or eliminate the proteinase K treatment. We use 4 ug/ml proteinase K for 3 min.

11) Stop the protease by washing 2x with PBT containing 2 mg/ml glycine.

12) Wash the embryos 2x with PBT.

13) Postfix for twenty min.

14) Wash the embryos 5x with PBT; 1x with 50% PBT: 50% hybridization buffer and once with hybridization buffer prior to placing them in 1 ml of hybridization buffer for 1 hr of prehybridization at 48oC***.
***Hybridizations with riboprobes are carried out at 55oC.

15) Add17 ng (17 ul) of heat-denatured probe to each tube containing 20-50 ul of embryos and 100 ul of hybridization buffer. Hybridize at 48oC (55oC for riboprobes) for 24-48 hrs.

16) Wash the embryos 4-5x in 1 ml of 48oC (55oC for riboprobes) hybridization buffer. For best results, the final wash should go overnight.

17) Rinse the embryos 1x with 50% hybridization buffer: 50% PBT, and 4x with room temperature (R.T.) PBT.

18) Incubate the embryos with 1 ml of a 1:2000 dilution of a preadsorbed alkaline phosphatase-conjugated anti-digoxigenin F'ab fragment (Boehringer-Mannheim Pharmaceuticals) for 2 hrs at R.T..

19) Wash away excess antibody with at least 10 changes of PBT over 2 hrs.

20) Equilibrate embryos with 2 changes of staining buffer.

21) Develop (usually for 1-2 hrs) in 1 ml of staining buffer to which 4.5 ul of NBT and 3.5 ul of X-phosphate have been added. Occasionally, to detect poorly transcribed messages, we develop overnight at 4oCwith 1 or 2 changes of developing solution.

22) Stop the developing by washing 5x with PBT + 50 mM EDTA.

23) Clear the embryos overnight at 4oC in10-90% glycerol, 50 mM EDTA. Mount embryos on slides in the same.

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