1. Linearize 10-20µg of plasmid to completion (check small aliquot on a gel).
2. Adjust volume to 100 µl with water, add 1 µl 0.5 M EDTA and 1 µl of proteinase K (10 µg/ml). Incubate several hours at 37°C.
3. Phenol extract and ethanol precipitate.
4. Spin, wash and dissolve pellet at appr. 1 µg/µl in water (check small aliquot on gel).
5. Transcribe 1 µg of template using Boehringer Mannheim Genius kit #4.
6. Stop reaction with 1 µl 0.5 M EDTA, add 2 µl 5 M LiCl and 75 µl EtOH and precipitate (-20°C over night or several hours on dry ice).
7. Spin, wash and dissolve pellet in 25 µl H ₂ O. Remove 1 µl to check amount and length of transcripts on a TAE gel. Use control RNA from kit to estimate RNA yield. In general, 5 to 20 µg of RNA will be produced. [At this point transcripts longer than 500 nucleotides should be hydrolized: Add equal volume of NaCO ₃ pH 10.2 (60mM Na ₂ CO ₃ /40mM NaHCO ₃ ) and incubate at 65°C (duration depends on length, e.g., 20 min for 1 kb). (Check small aliquot together with unhydrolized sample from step 7 on a TAE gel.) Stop hydrolysis by adding equal volume of 0.2 M NaAc pH 6. Add 3 volumes of EtOH and precipitate as above.] If proceeding without hydrolysis add 2 µl 5M LiCl and 3 volumes of EtOH and precipitate.
8. Spin, wash and dissolve pellet in 100 µl hybridization mix (final concentration 0.05 to 0.2 µg/µl). Probes are stable for months at -20°C.

1. Remove paraffin in xylene (2 x 10 min).
2. Rehydrate 2 min each in 100 %, 95%, 70%, 50% EtOH.
3. Place at 65°C in 2xSSC for 30 min (removes residual paraffin, xylene).
4. Cool down slowly by adding distilled water.
5. Digest 10 min with proteinase K (2 µg/ml in 50 mM Tris pH 7.5; 5 mM EDTA) at r.t.
6. Stop protease by placing slides for several min in 2 mg/ml glycine in PBS.
7. Post fix with 4% paraformaldehyde in PBS for 10 min.
8. Wash several times in PBS.
9. Acetylation: Put 250 ml 0.1 mM triethanolamine pH 8 (3.4 ml triethanolamine/250 ml H₂O, add 525 µl conc. HCl) in a staining dish equipped with a stirring bar. Place slides in a slide rack in the dish (use short pieces of a pipette as spacer to allow for the stir bar) and slowly add 675 µl acetic anhydride with mixing. Incubate for 10 min.
10. Rinse several times in PBS.
11. Dip briefly in H₂O. Flick off excess water and proceed to hybridization.

Depending on the efficiency of transcription, I use 1-2 µl digoxigenin-labeled probe diluted in 30 µl hybridization mix per slide.

1. Drop 20-30 µl hybridization mix containing the probe on a cover glass that is slightly larger than the area of the sections. Invert slide onto drop of probe and pick up cover glass.
2. Seal cover glass with rubber cement, place slides in a slide box containing wet paper towel. Incubate horizontally at 50OC over night.

1. Carefully remove rubber cement seal and place slides several min in 2xSS until cover glass falls off.
2. Wash 1x 15 min in 50% formamide, 2xSSC at 65°C.
3. Wash 1x 30 min in 50% formamide, 2xSSC at 65°C.
4. Wash 2x 30 min in 25% formamide, 2xSSC at 65°C.
5. Wash 1x 5 min in 2xSSC at r.t.
6. PLACE SLIDES INTO CUVETTES THAT ARE RESERVED FOR RNASE TREATMENT ONLY!!! (To avoid RNAse contamination of all your glassware.)
7. Incubate with 5 µg/ml RNAse A in 2xSSC for 15 min at r.t.
8. Wash several times in PBS.
9. Incubate with 1% blocking agent in maleate buffer (100 mM maleic acid; 150 mM NaCl, adjusted to pH 7.5) for 30 min to 1 hour at r.t. (Blocking agent from Boehringer Mannheim Genius kit #3 has to be prepared in advance by heating. It does not dissolve completely but stays somewhat cloudy.)
10. Incubate with antibody diluted 1:500 in 1% blocking agent in maleate buffer for 1 hour.
11. Incubate 2x 5 min in NMBT (100 mM Tris pH 9.5; 100 mM NaCl; 50 mM MgCl2; 0.1% Tween 20; make fresh from stocks before use).
12. Perform color reaction by incubating in NMBT containg 3.5 µl/ml BCIP (bromo-chloro-indolyl-phosphate; 50 mg/ml in dimethylformamide) and 4.5 µl/ml NBT (nitro-blue-tetrazolium; 75 mg/ml in 70% dimethylformamide. Either from Genius kit or prepared from powder. Use fresh DMF or filter!) Keep the reaction in the dark. Check intensity of color development from time to time. (Some light does not seem to harm.) Signals for milk proteins (very abundant) appear within 1 hour; less abundant messages take over night to 1 day.
13. When color is strong enough, wash slides in PBS; quickly rinse in water, counterstain (e.g., Nuclear Fast Red), dehydrate and mount.  

    Hybridization mix:

    • 50% formamide
    • 5x SSC
    • 0.1 M Na-phosphate buffer pH 7
    • 1x Denhardt's solution
    • 100 mM DTT
    • 100 µg/ml carrier RNA (E. coli or yeast)
    • 100 µg/ml sonicated salmon sperm DNA