In situ hybridization experiments on frozen sections
In situ hybridization experiments on frozen sections
In situ hybridization of cellular RNA is used to localize specific mRNAs in mixed cell populations and tissues. Frozen section is a method of slicing tissue after it has been rapidly cooled to a certain hardness at low temperatures. Because it is faster and easier than paraffin section, it is mostly used for rapid pathologic diagnosis during surgery.
Operation method
basic program
Materials and Instruments
Frozen Sections Move 1. Remove slides from the refrigerator, equilibrate to room temperature, and open the slide box. Place the slides in a rack and soak them in streptavidin solution prepared with 50 mmol/l Tris-Cl (pH 7.5)/5 mmol/l EDTA for 10 min. For more product details, please visit Aladdin Scientific website.
Tris-Cl EDTA Glycine PBS SSC Ethanol Formamide Dextran Sulfate DTT NaCl
Humidifier Water Bath Incubator
2. Immerse the slide in PBS containing 2 mg/ml glycine for 30 s at room temperature, then in PBS twice for 30 s each.3. Immerse the slide in freshly prepared TEA buffer for 5 min.4. In a beaker, dispense enough TEA buffer to cover the slide. Add liver acetate to a final concentration of 0.25%, pour quickly onto the slide, shake the slide holder to mix the liquid well, and leave for 10 min.
5. Wash the slides twice in 2× SSC for 5 min each time.6. Aman was dehydrated by sequential soaking in 30%, 60%, 80%, 95% and 100% ethanol for 2 min each time, dried, sectioned and used immediately for hybridization.7. Precipitate the labeled DNA or RNA probe. Determine the percentage of label incorporation (specific activity) and estimate the amount of probe synthesized, and resuspend the probe precipitation accordingly by estimating the final volume of hybridization mix at a final concentration of 0.2 μg/ml per kilobase.8. The probe precipitate is first resuspended with 2 parts of Hybridization Mix B and 2 parts of deionized formamide, followed by the addition of 1 part of 50% dextran sulfate and mixing thoroughly.9. Boil labeled DNA probes for 2 min and place immediately in an ice bath; or heat labeled RNA probes at 80°C for 30 s and maintain at 50°C. After heat denaturation, add 3.3 mol/l DTT to 50 mmol/l final concentration. Using a spiking tip, add enough probe to cover the section well.
10. The slides were incubated for 4 h in a well-closed humidified box filled with humidified box solution B. The probes were incubated at 37 ℃ and the probes were incubated at 42 ℃.
11. For DNA probes: Wash with DNA wash solution preheated to 37 ℃ for 2 h, and change the wash solution 4~5 times in the meantime.
12. For RNA probes:(1) Wash in RNA Wash 1 preheated to 50°C for 15 min at least twice.(2) Slides were incubated for 15 min at 37°C in 0.5 mol/l NaCl/10 mmol/l Tris-Cl (pH 8.0) solution containing 20 μg/ml of boiled RNAase A. The slides were incubated for 15 min at 37°C in 0.5 mol/l NaCl/10 mmol/l Tris-Cl (pH 8.0) solution.
(3) Sin 15 min in RNA Wash 1 preheated to 50°C for at least 2 washes.
(4) Wash in RNA Wash 2 preheated to 50°C for 15 min and change the solution twice. (The wash temperature can be increased to 60°C if necessary, but this may affect the morphological results.)13. Dehydrate by immersion for 2 min each in the following solutions: 30% ethanol with 0.6 mol/l NaCl, 60% ethanol with 0.6 mol/l NaCl, 80% ethanol, 95% ethanol and 100% ethanol.
14. After drying the sections in air, the probes for hybridization are detected by autoradiography.