In Situ Hybridization Of Specimens

Once treatment of specimens with DNase or RNase and preparation of a specific probe are complete, one can proceed to in situ hybridization. In general, the procedure for DNA hybridization is similar to that of RNA hybridization except for differences indicated at specific steps.

1. Carry out prehybridization as follows:

a. Carefully overlay tissue specimens with an appropriate volume of pre-hybridization solution (15 to 20 ml/10 mm2) using the tip of a pipette or a plastic coverslip cut slightly larger than the tissue section.

b. Place the slides in a moist chamber and allow prehybridization to proceed for 1 h at 37°C.

c. During prehybridization, prepare hybridization cocktail (see Reagents Needed).

d. After prehybridization is complete, carefully remove the prehybridiza-tion solution from the slides with a 3MM Whatman paper strip or the tip of a pipette.

2. Perform hybridization and washing as follows:

a. Immediately overlay each specimen with freshly prepared hybridization cocktail containing the specific probe (15 to 20 ml/10 mm2). Cover the cocktail with a plastic coverslip cut slightly larger than the tissue section.

b. Simultaneously denature both probe and target DNA or RNA by placing the slides on a hot plate or an equivalent for 3 to 4 min at 95 to 100°C for DNA hybridization or at 55 to 65°C for RNA hybridization.

Notes: (1) DNA or RNA in the specimens can be denatured prior to prehybridization. (2) Labeled probes can be denatured immediately before preparation of the hybridization cocktail. (3) The concentration of the probe is critical for the success of in situ hybridization using a nonra-dioactive probe. A concentration of 2 mg/ml DNA probe or 1 mg/ml RNA probe is recommended. The efficiency of RNA probes is higher than that of DNA probes because some of the denatured ssDNA probes can be renatured to dsDNA under hybridization conditions. If a high background occurs, the concentration of the probe can be reduced 5- to 10fold.

c. Quickly place the slides in a high-humidity chamber and carry out hybridization at 37°C for 2 to 4 h.

Notes: (1) Any bubbles underneath the plastic coverslips should be removed by gently pressing the coverslip or by using a toothpick. (2) In order to prevent uneven hybridization, slides should be kept level in the chamber. (3) The humidity chamber must be tightly covered or sealed prior to being placed in a hybridization oven or an equivalent. If the specimens are dried during hybridization, a high background will occur. (4) The relatively high concentration of formamide (50%) and low salt concentration (e.g., 2X SSC) in the hybridization cocktail solution are essential and have two functions in general. One is to facilitate denaturing of target DNA or RNA and probe at 100° C, about 35 to 40°C above the Tm of homologous hybridized DNA. The other is to prevent potentially nonspecific binding of the probe while not interfering with probe-target annealing at 100°C. (5) Inasmuch as RNA-RNA hybrids have a relatively higher Tm than DNA-DNA hybrids, it is recommended that, prior to hybridization, the RNA probe and the target RNA be heated at 65 to 70°C to denature any secondary structure in RNA molecules. In addition, hybridization temperature should be set up at 45 to 50°C for RNA-mRNA in situ hybridization, and 40 to 45°C for DNA-RNA in situ hybridization.

d. Following hybridization, quickly place slides in a slide rack and insert the rack into a staining dish filled with washing solution containing 2X SSC and 2.5% BSA. Wash the slides with three changes of fresh washing solution, 15 min each at 45°C with gentle shaking.

e. Continue to wash the slides twice, 5 min each time, in fresh washing solution containing 0.2X SSC, 2.5% BSA and 0.05% Tween 20 in PBS at room temperature with gentle shaking.

Note: Do not allow the specimens to become dry prior to detection. At this stage, unhybridized RNA of the tissue specimens can be removed using an appropriate amount of RNase (DNase free) or a mixture containing 45 mg/ml RNase A, 2 fg/ml RNase T1 (Sigma Chemicals), 5 mM EDTA and 250 mM NaCl in TE buffer, pH 7.5. RNase treatment could enhance the signal-to-noise ratio, reducing the background.


1. Overlay the specimens with an appropriate volume of streptavidin-alka-line phosphatase (AP) conjugate solution (for biotin probes) or anti-DIG-alkaline phosphatase solution (for DIG probes) at an appropriate dilution.

2. Place the slides in an aluminum foil-wrapped high-humidity chamber and incubate for 20 to 30 min at 37°C or 1 h at room temperature.

3. Carefully remove the solution from the specimens and carry out washing in PBS containing 0.05% Tween 20 in a slide dish for 3 x 5 min at 42°C with gentle agitation.

4. Place the slides in a slide rack and wash them in an appropriate volume of enzymatic predetection solution for 2.5 min.

5. Replace predetection solution with fresh enzymatic detection solution containing NBT and BCIP as substrates.

6. Place the slides in a container and wrap it with aluminum foil. Allow color to develop at room temperature for 20 to 120 min. Check results once every 4 min under a microscope.

Note: Color development should be time controlled. Overdevelopment may bring up higher background. For this reason, it is recommended to stop the color development once the primary color is visible, assuming that the probe-target binding that results in the strongest signal is the primary color.

7. Wash the slides in PBS for 3 x 3 min and counterstain the specimens in nuclear fast red solution for 5 min if desired.

Note: Nuclear staining is omitted for RNA-RNA in situ hybridization if the specimens are pretreated with DNase I.

8. Partially dry the slides and mount the specimens with glass coverslips using Permount™.

9. View and photograph with a phase-contrast microscope.

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