Detection Of Hybridized Signals

For large organs or tissues, tape the slides to a film exposure cassette or cardboard and expose them against the film at 4°C for 1 to 10 days prior to emulsion autora-diography. In general, slides are directly subjected to autoradiographic emulsion by the following method:

1. Add an appropriate volume of working NTB-2 emulsion (prethawed in a 45°C water bath) to a slide-dipping chamber.

2. Slowly and carefully dip the slides into the dipping chamber and gently pull them out. Place the slides vertically in an appropriate rack and air-dry them for 2 h or in front of a fan for 20 min.

3. Place the slides in a light-tight slide box containing desiccant and seal the box with black tape. Thoroughly wrap the box with aluminum foil and allow exposure to proceed at 4°C for 1 to 10 days, depending on the intensity of the signal.

Notes: (1) The exposure of slides mandates a-32P-free environment. Thus, make sure to perform a radioactive survey of the refrigerator or cold room where the exposure takes place. (2) The slides must be completely dried prior to exposure because moisture results in fading of the images in the autoradiographic emulsion.

4. In a darkroom with a safe light, add sufficient volumes of developer, water, and fixer to slide jars or staining dishes and allow the solution and water to warm up to 20°C.

5. Transfer the slide exposure box from the refrigerator or cold room to the darkroom and allow the box to warm to 20°C.

6. Turn on an appropriate safe light in a light-tight darkroom, open the exposure box and place the slides in a slide rack.

7. Carefully immerse the slides in developer for 2 to 4 min; quickly rinse the slides in water for 30 s and immediately place the slides in fixer for 3 min.

8. Wash the slides in slowly running tap water for 30 min. Turn on the normal light, remove the emulsion on the back side of the slide using a razor blade and air-dry the slides in a dust-free place.

9. Carry out slight counterstaining of the slides in order to enhance the images using the hematoxylin/eosin-staining procedure described next:

a. Prepare a series of solutions in slide-staining dishes in the order below: 1 dish: hematoxylin stain; 2 dishes: water; 1 dish: 0.1% NH4OH; 2 dishes: water; 1 dish: eosin stain; 1 dish: 95% ethanol; 3 dishes: 100% eth-anol; 3 dishes: xylene b. Prewet the developed slides in water in a staining dish for 2 min.

c. Stain the specimens in hematoxylin for 10 to 30 sec and rinse the slides in water for 2 x 2 min.

d. Quickly dip the slides in 0.1% NH4OH and rinse in water for 2 x 3 min.

e. Stain the specimens in eosin 10 to 30 sec and quickly dip the slides nine times in 95% ethanol, then nine times in 100% ethanol.

f. Dehydrate the specimens for 2 min in 100% ethanol, then 3 min in fresh 100% ethanol, followed by 2 x 2 min in xylene g. Place the slides flat and add a few drops of PermountTM to one end of the specimens and slowly overlay them with a clean coverslip. Carefully remove excess Permount with 3MM Whatman paper filter strips.

h. Seal the edges of the coverslip with melted wax or allow the slides to air-dry for 1 to 2 days.

i. Observe and photograph the specimens using darkfield microscopy according to the manufacturer's instructions.

Reagents Needed

5X Labeling Buffer

25 mM MgCl2

26 A260 units/ml random hexadeoxyribonucleotides

5X Transcription Buffer 30 mM MgCl2 0.2 M Tris-HC, pH 7.5 10 mM Spermidine 50 mM NaCl

NTPs Stock Solution

100 mM ATP in dd.H20, pH 7.0 10 mM GTP in dd.H20, pH 7.0 10 mM UTP in dd.H20, pH 7.0 10 mM CTP in dd.H20, pH 7.0

Specimen Blocking Solution

Warm an appropriate volume of PBS to 45°C, add 0.16% (w/v) DTT, 0.19% (w/v) iodoacetamide, and 0.13% (w/v) N-ethylmaleimide. Mix well and cover with aluminum foil prior to use.

Note: This solution should be freshly prepared and used immediately. Because it contains toxic iodoacetamide and N-ethylmaleimide, gloves should be worn and caution should be taken.

PBS Buffer

2.7 mM KCl 1.5 mM KH2PO4 136.9 mM NaCl 15 mM Na2HPO4 Adjust the pH to 7.3.

20X SSC Solution

0.3 M Na3Citrate (trisodium citric acid) 3 M NaCl

Adjust the pH to 7.0 with HCl; autoclave.

50X Denhardt's Solution:

1% (w/v) BSA (bovine serum albumin) 1% (w/v) Ficoll (Type 400, Pharmacia) 1% (w/v) PVP (polyvinylpyrrolidone)

Dissolve well after each addition; adjust to the final volume with distilled water and sterile-filter. Divide the solution into 50-ml aliquots and store at -20°C. Dilute 10-fold prior to use.

Prehybridization Solution

50% (v/v) Deionized formamide 15 mM Tris-HCl, pH 8.0 2 mM EDTA, pH 8.0 2X SSC

5X Denhardt's solution

0.2% Denatured and sheared salmon sperm DNA for DNA hybridization or 0.2 mg/ml yeast tRNA and 0.2 mg/ml poly(A) for RNA hybridization.

Hybridization Cocktail

Add [35S]dCTP- or [35S]UTP -labeled probe to an appropriate volume of fresh prehybridization solution.

Triethanolamine (TEA) Buffer

Working Emulsion Solution

Thaw Kodak NTB-2 autoradiographic emulsion at 45°C and warm an equal volume of dd.H2O at the same temperature. Gently mix together Kodak NTB-2 solution and water, and wrap the container containing the working emulsion solution with aluminum foil prior to use.


Developer Fixer

Hematoxylin stain

0.1% NH4OH (freshly prepared)

Eosin stain

DNase I (RNase free)

RNase I (DNase free)

SP6, T7 or T3 RNA polymerase

15 mg/ml Yeast tRNA or poly(A) RNA as a carrier

0.5 M Dithiothreitol (DTT)

2 M NaCl solution

7.5 M Ammonium acetate solution

TE-saturated phenol/chloroform

Chloroform:isoamyl alcohol: 24:1 (v/v)

Deionized formamide, ultrapure grade

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