In Situ Hybridization

experimental steps

1. Preparation of RNase-free glassware and solutions 2. Tissue preparation by the standard histological method 3. Preparation of the probes 4. Probe hydrolyzation 5. Tissue pretreatment 6. Nucleic acid hybridization 7. detection

7. detection

By autoradiography for 35S-labeled probes: using emulsion that produces silver grains that are viewed in dark-field microscopy. By immunolocalization for digoxigenin-labeled probes: using anti-digoxigenin antibody conjugated with alkaline phosphotase that catalyzes NBT-BCIP to form a brown/blue precipitate at the site of RNA localization.

6. nucleic acid hybridization

Calculate the amount of working probe solution - 100-200 µl/slide. Denature probe at 80°C, chill it on ice, and properly dilute the probe in hybridization buffer. Apply probe to the sections, cover the slides with coverslip or another slide, and incubate in a closed moist chamber at 50°C overnight. Washes and RNase treatment (remove single-stranded RNA molecules), and more washes

2. tissue prep by standard hist method

Fix the tissue in a fixative. Dehydrate the tissue. Use paraffin-embedding. Mount sections on Probe-On Plus or equivalent slides to achieve the best section retention.

4. probe hydrolyzation

Full-length probes if much longer than 300 base pairs (bp) won't penetrate the fixed cytoplasm. Hydrolyze the probe to a length of approximately 150-300 bp. Probes <100 are too small for specific hybridization with the target mRNA. The size range and yield of the hydrolyzed probe should bedetermined.

3. prep of probes

Linearize the template (plasmid) DNA by cutting with a single restriction enzyme. Synthesize (transcribe) either the sense or antisense RNA probes in a buffer containing the unlabeled ATP, GTP, CTP, labeled UTP (35S or digoxigenin), the linearized DNA, and the appropriate RNA polymerase; the sense probe can be used as a negative control .

1. prep of RNase-free glassware and solns.

RNase contamination can degrade the mRNA that is under investigation. Bake glassware to kill RNase, and use DEPC-treated DI to prepare solutions to inactivate RNase in the solutions.

4. tissue pretreatment

Remove paraffin with 3 fresh xylene solutions. Hydrate sections. Proteinase K treatment - make crosslinked fixed protein matrix permeable to probe. Formaldehyde postfixation - stabilize proteinase-treated tissues. Acetic anhydride treatment - reduce electrostatic binding of probe. Dehydrate and air dry the tissue sections. Optional prehybridization in hybridization solution minus probe - reduce background signal.

in situ hybridization

The methods used to localize mRNA or single-stranded (ss) DNA at the tissue or cellular level. Labeled ssDNA or ssRNA probes are used to hybridize with in vivo mRNA or DNA that is denatured to become ssDNA prior to hybridization. It is based on the property of RNA and ssDNA that two single-stranded such molecules form a double-stranded molecule if they have complementary sequences. The hybridization can occurbetween RNA and RNA, DNA and RNA, or DNA and DNA.