In situ hybridization experiments for cellular RNA are used to localize specific mRNAs in mixed cell populations and tissues.
Operation method
basic program
Materials and Instruments
Paraffin Sections Move 1. Prepare the dewaxing/rehydration system (reusable several times) and 0.2 mol/l HCl while the slides containing the sectioned samples (in slide boxes, stored at -20°C or -70°C) are brought to room temperature. Begin preheating 2 × SSC to 70°C (used in step 5).2. Deparaffinize sections in staining trays with xylene, replacing xylene three times at 2-min intervals (not necessary for slides containing unembedded single cells). Caveat 1. All of the above steps are performed by placing a slide bearing the sample in a slide holder and in a glass staining dish containing the indicated solutions, all of which are freshly prepared and used only once unless otherwise indicated. 2. iodoacetamide and N-ethylmaleimide are highly toxic and should be handled with care. For more product details, please visit Aladdin Scientific website.
HCl Xylene Ethanol SSC PBS Streptavidin Glycine DTT RNAase Digest Ammonium Acetate
Staining trays Humidifying boxes Slides Ovens Water baths Incubators
3. Rehydration was carried out through the following groups of stained discs: 100% ethanol twice for 2 min each; 95% ethanol for 2 min; 70% ethanol for 2 min and 50% ethanol for 2 min.
4. Samples were denatured in 0.2 mol/l HCl for 20 min at room temperature.
5. Samples were heat denatured in 2× SSC at 70°C for 15 min, immersed in 1× PBS for 2 min.6. The samples were post-fixed in freshly prepared 4% PFA fixative and left at room temperature for 5 min. Fixation was terminated in 3×PBS for 3 min, followed by 2 immersions in 1×PBS for 30 s each.
7. Equilibrate the samples in 1 x PBS containing 10 mmol/l DTT and leave at 45°C for 10 min.8. Seal with freshly prepared blocking solution. Place in a water bath at 45°C for 30 min and cover the water bath with aluminum foil (iodoacetamide is light sensitive).9. At room temperature, wash twice with 1× PBS for 2 min each.10. Equilibrate the samples in freshly prepared TEA buffer for 2 min, mount the slides in fresh TEA buffer and add acetic anhydride to 0.25% final concentration. Mix rapidly and soak slides for 5 min with agitation, add acetic anhydride to 0.5% final concentration and soak for another 5 min.
11. Samples were transferred to 2 x SSC for 5 min.12. Sample dehydration was carried out by immersion in 50% ethanol, 70% ethanol, 95% ethanol and 100% ethanol (2 times). 2 min each at room temperature (with step 3 ethanol solution).
13. Air-dried samples (or dried in a desiccator) should be certified as absolutely dry before proceeding with the experiment, and the samples should be placed in desiccant-filled slide boxes and stored dry at -70°C overnight.
14. Centrifuge ethanol-precipitated reverse- and forward-stranded 35S-labeled nucleic acid probes as well as S-ribonucleic acid probe competitors. After the precipitate dries, dissolve the precipitate with 5 μl of sterile 5 mmol/l DTT. Add 2.5 μl (half of the reaction) of S-ribonucleic acid probe competitor to the reverse and forward chain ribonucleic acid probes.
15. Denature the probes by heating at 100°C for 3 min.
16. Immediately add sufficient Hybridization Solution A to give a final probe concentration of 0.3 μg/ml and mix thoroughly. Determine the radioactivity (expect a radioactivity count value of ≥1 x 105 cpm/μl). Place the tube in a 45°C water bath (hybridization temperature).17. Carefully spread the appropriate amount of probe on the specimen (if using a spiking tip, spread at 20 μl/20 mm3 to start hybridization).18. Place the samples in a humidor (slides placed horizontally), add humidor solution A to the humidor, and incubate the hybridization for an appropriate period of time at 45℃. Hybridization can be carried out for 30 min~4 h. (The liquid in the humidor must be equilibrated and the humidor must be carefully sealed because if the sample dries out, it will result in the appearance of a high hybridization background, and the osmolality of the humidor solution and the hybridization mixture must be the same in order to prevent the hybridization solution from diluting or concentrating).
19. Prepare and preheat Wash A, B, and C during the last hour of the hybridization process.
20. Begin slide washing by immersing slides in 55 °C 100 ml Wash A. Immediately place in a slide holder and into a staining tray containing Solution A.21. Wash 2 times for 15 min each in Solution A at 55 °C. Continue with 2 washes for 15 min each in Solution B at 55 °C. Then, wash 2 times for 2 min each in Solution C at room temperature.
Figure I. Hybridization mixture on slides
22. Add 500 μl of RNAase digestion solution to each slide, covering all of the sample, and place the slides in a humidified box (with water) labeled for RNAase. Leave at room temperature for 15 min.
23. Wash the slides twice for 30 min at 50°C in Wash C. Wash the slides twice for 30 min at 50°C in Wash A. Continue to wash the slides twice for 5 min each at room temperature with 2×SSC.
24. Dehydration was carried out in the following groups of liquids (2 min each): 50% ethanol/0.3 mol/l ammonium acetate, 70% ethanol/0.3 mol/l ammonium acetate, 90% ethanol/0.3 mol/l ammonium acetate, 100% ethanol.
25. Dry the slides in air. Expose the film to pressure at least overnight, followed by latex radiation autoradiography.