Isotope-labeled probes were hybridized with cells fixed on microscope slides, and then the hybridization sites were visualized by radiographic autoradiography.
Operation method
Program 27.8 Radiation autoradiography in in situ hybridization
Principle
Isotope-labeled probes were hybridized with cells fixed on microscope slides, and then the hybridization sites were visualized by radiographic autoradiography.
Materials and Instruments
Glycogen Agarose Dithiothreitol Dithiothreitol Move RNA preparation For more product details, please visit Aladdin Scientific website.
Diethyl pyrocarbonate DEPC-water Phenol-chloroform-isoamyl alcohol Sodium acetate Anhydrous ethanol Chromatography column mobile phase buffer Phenol-chloroform-boundary amyl alcohol Scintillation solution Tissue washing solution NaCl-DEPC EDTA Proteinase K buffer Proteinase K storage solution Formaldehyde Triethanolamine Glacial acetic acid DTT Hybridization solution Gelatin RNaseA storage solution RNase buffer β-mercaptoethanol 50% formaldehyde SSC
Microcentrifuge Tubes Microcentrifuge RNA Labeling Kit G50 Dextran Gel Chromatography Columns
All solutions used in the preparation of the probes, as well as the eluent after hybridization, must be free of RNAase. The solutions are DEPC-treated and autoclaved for 20 min at 121°C. Although this process removes most of the RNAase, because of the widespread presence of natural RNAase, even careful handling cannot guarantee that RNAase residues on glassware and pipette tips will be completely removed. Therefore, for RNA manipulation, a separate set of pipette tips is required, which are routinely soaked overnight in DEPC water or treated with an appropriate RNAase inhibitor, such as RNase-Zap (Ambion); all glassware should be wrapped in aluminum foil and autoclaved prior to use; and Eppendorf centrifuge tubes should be treated with either DEPC water or RNase-Zap and then autoclaved. Eppendorf tubes should be treated with DEPC water or RNase-Zap and then autoclaved.
Probe Preparation
RNA probes (ribosomal probes)
To prepare RNA probes, a DNA strand corresponding to the target sequence is used as a template, which is doped with radioactive nucleotides to generate positive and negative RNA probes.
If high activity probes are required, the ideal choice is single-stranded RNA probes; usually the length of the probe is 200~1000 base pairs (bp), but the optimal length is 150~200 bp, because the longer the labeled probe is, the weaker its tissue penetration. If desired, the probe length can be shortened by limited alkaline hydrolysis; RNA-RNA or RNA-DNA hybrids are more stable than oligonucleotide-oligonucleotide and DNA-DNA hybrids, so the first two are most commonly used as probes.
Commercial Probes and Control Probes
Commercial DNA templates for actin and GAPDH can be used to prepare RNA probes, and are often used as positive controls because they are housekeeping genes and are commonly expressed in eukaryotic cells. Negative controls are often used as positive strand probes, and the use of negative controls can exclude false positives and produce more accurate results. For each hybridization, a tumor specimen with a clear tissue type and a specific RNA expression profile should be loaded on the slide as a positive control for objective evaluation of the hybridization results. These positive control specimens are commercially available from the relevant biological companies.
Linearization of Plasmid DNA
1. Add 10-20 μg of DNA to each microcentrifuge tube.
2. Add 10 units of restriction endonuclease to each μg of DNA and digest according to the instructions.
3. Digest at 37°C for 3 h or overnight.
Phenol/Chloroform Extraction
4. Add 400 μl of phenol-chloroform-isoamyl alcohol (pH 8.0) and mix with vortexing.
5. Centrifuge at 13,000 rpm for 3 min at room temperature.
6. Collect the supernatant, transfer it to a new tube and add 10 μl of NaAc (3 mol/L, pH 8).
7. Add 250 μl of 100% ethanol (stored at -20°C).
8. Add 1 μl of glycogen to promote DNA precipitation.
9. Place the centrifuge tube on dry ice for 1 hour.
10. Centrifuge at 13,000 rpm for 15 min.
11. Discard the supernatant and retain the precipitate.
12. Add 400 μl of 70% ethanol (stored at -20°C) to wash the precipitate.
13. Centrifuge at 13,000 rpm for 10 min.
14. Discard 70% ethanol and allow the precipitate to dry at room temperature.
15. Depending on the amount of DNA used, re-dissolve in 10-20 μl of DEPC water (see step 1) to a final concentration of 1 μg DNA/μl.
16. Take 0.5 μl of DNA sample solution and electrophoresis with 1% agarose gel.
RNA labeling
For procedures involving the incorporation of radioactive nucleotides, refer to the instructions accompanying the Amersham kit (RPN3100; Amersham Biosciences ), which are described below:
1. Mix the following components in a microcentrifuge tube
(a) 4 μl of 5× Transcription Buffer.
(b) 1 μl 0.2 mol/L DTT.
(c) 1 μl HPRI.
(d) 0.5 μl ATP, CTP and GTP.
(e) 1 μl linear DNA template (1 μg/ml ).
(f) 9.5 μl 35S-UTP.
(g) 2 μl RNA polymerase.
2. Mix the components well and place the solution at 37°C for 1.5 h
DNA Enzymatic Extraction of DNA Templates
3. Add 10 U DNase I.
4. Add 1 μl of RNase inhibitor.
5. Mix the solution well and place at 37°C for 10 min.
Removal of Undoped Nucleotides
6. Equilibrate the G50 dextran column with 2 ml of column buffer.
7. Load the probe onto the column.
8. Add 400 μl of column buffer to elute, let stand to allow all eluate to drain off, discard.
9. Add another 400 μl of column buffer and collect the eluate in an Eppendorf tube.
Phenol/chloroform extraction
10. Add 400 μl of phenol (pH 5.0) to the collection solution of the Eppendorf tube, mix by vortexing and centrifuge at 13,000 rpm for 3 min.
11. Collect the supernatant, transfer to a new microcentrifuge tube, add 400 μl of chloroform-isoamyl alcohol, and centrifuge at 13,000 rpm for 3 min with vortexing and mixing.
12. Collect the supernatant and take 1 μl of the supernatant to determine the amount of incorporation.
13. Add 2.5 times the volume of frozen 100% ethanol at -20℃ to the remaining supernatant.
14. Add 1 μl of yeast glycogen to promote precipitation.
15. Allow the Eppendorf tube to dry in air for 30 min.
16. Centrifuge at 13,000 rpm for 15 min.
17. Pipette off residual ethanol, do not touch the precipitate.
18. Wash the precipitate with 70% ethanol and centrifuge at 13,000 rpm for 10 min, then discard the ethanol.
19. Allow the precipitate to dry and re-suspend with 50 mmol/L DTT (to make a suspension), calculating the volume of 50 mmol/L DTT to be added by the following method:
(a) Take 1 μl of the supernatant obtained in step 12, add 2~3 ml of scintillation solution and measure the number of scintillations per minute (CPM ) on a liquid flash counter.
(b) DTT volume = CPM × 400 ÷ (6 × 105 )
The 400 in the formula is the volume after the addition of phenol/chloroform extract, and 6 × 105 is the total CPM value measured in the DTT solution.
(c) This gives the volume to be added to 50 mmol/L DTT.
20. Remove 1 μl of the solution and count the CPM again to confirm the specific activity of the probe.
In situ hybridization
Preparation of specimens
The following procedure is designed to maintain the structural and morphological integrity of the tissue without destroying cellular RNA. RNA degradation can be avoided by treating the tissue samples with methods such as rapid freezing or formalin fixation. Crosslinking fixatives such as 4 % paraformaldehyde and 4 % formaldehyde are preferred to preserve RNA without affecting its specific binding. The duration of tissue fixation depends on the size of the sample. The longer the fixation time, the better the morphology of the tissue will be maintained, but this will also reduce the binding to the probe. Paraffin can be used as an embedding agent, and paraffin-embedded specimens can be sliced to a thickness of 1 μm and easily deparaffinized prior to hybridization. Since paraffin sections require a variety of subsequent treatments in various solutions, it is recommended that a coated slide be used to ensure that the sample does not slip. Frozen samples should be cooled to -70°C before sectioning, and frozen sections should be placed on coverslips for fixation.
Tissue preparation prior to hybridization
Prior to hybridization, the following treatments are performed to increase the affinity of the RNA target sequence for the probe and to reduce non-specific background. The affinity of the target nucleotide for the probe is increased in protease-treated samples, especially if the probe length is greater than 100 bp. In order to inhibit tissue metamorphosis, it is important to post-fix the samples with formaldehyde. Application of glacial acetic acid for acetylation reduces the non-specific background due to binding to the amino terminus of the protein. During tissue preparation, high priority is given to the degrading effect of RNA enzymes. To remove any possible contamination, all glassware and solutions must be sterilized and DEPC'd separately.
Gloves must be worn throughout the procedure. The opportunity to touch the tissue sections should be minimized.
Hybridization
Hybridization temperature is critical for stable binding of certain probe/target sequences. Although formalin is a de-stabilizer of helical chains, the addition of formalin to the hybridization buffer will lower the unlinking temperature of the hybridized chains, making it possible for the hybridization process to take place in a cooler temperature environment. The lower the hybridization temperature, the better for maintaining the structure of the tissue. To date, the optimal temperature has been found to be 52°C . The addition of dextran sulfate to the hybridization buffer reduces the hybridization volume, increases the probe concentration, and shortens the hybridization time. Although the hybridization reaction can be completed within 5-6 h, overnight hybridization is usually used. The addition of sodium ions to the hybridization buffer will stabilize the hybridization system.
Elution after hybridization
The main purpose of post hybridization elution is to remove unbound or non-specifically bound probes by selecting different temperatures, salt concentrations, and formamide concentrations. RNAase can be used to digest single-stranded RNA and unbound target sequences, but it does not degrade bound RNA-RNA complexes.
Preparation of Paraffin Sections
1. Paraffin sections are deparaffinized twice with Histoclear for 10 min.
2. rehydrate with graded concentrations (100%, 90%, 70%, 50%, 30%) of ethanol for 10 s each time.
3. 0.85% NaCl and D-PBSA solution were used for 5 min.
4. Place the tissue sections in 200 ml of Proteinase K buffer (diluted with 400 μl of Proteinase K stock solution) and digest for 7.5 min.
5. Wash with D-PBSA for 3 min.
6. Post-fix with 4% formaldehyde or 4% paraformaldehyde.
7. Drench with DEPO water for 1 min.
8. In a fume hood, acetylate the sections with 200 ml of triethanolamine (containing 500 μl of glacial acetic acid) at 0.1 mol/L for 10 min with constant and thorough stirring.
9. The sections were washed with D-PBSA and 0.85% NaCl for 5 min.
10. The sections were dehydrated in graded concentrations (30%, 50%, 70%, 90%, 100%) of ethanol for 10 s each.
11. Air-dry the sections.
Probe preparation and hybridization
12. Take 16 μl of 1 mol/L DTT, 344 μl of 60% hybridization solution (Hybridmix) and 40 μl of probe, mix them, vortex them and centrifuge them for a few moments, the mixture can be used to hybridize 20 paraffin sections.
13. Denature the probe at 80°C for 3 min, then cool on ice.
14. Take 20 μl of the probe mixture from step 12 and drop onto each of the 20 tissue sections, and cover the sections with a glass coverslip.
15. Hybridize overnight at 52°C in a humidity-saturated incubator.
Elution after hybridization
16. Preheat the eluent to the desired temperature.
17. Elute the sections with 200 ml of eluent (5×SSC containing 250 μl of β-mercaptoethanol) at 50°C for 30 min.
18. At 65°C, rinse the sections with 200 ml of eluent (2×SSC containing 50% formamide and 1.4 ml of β-mercaptoethanol) for 20 min.
19. At 37°C, elute twice with 200 ml of RNase buffer for 10 min each time.
20. 30 min at 37°C with 200 ml RNaseA buffer (containing 400 μl RNaseA).
21. Repeat step 19 for 15 min each rinse.
22. Repeat step 18.
23. At 50°C, rinse with 200 ml of 5×SSC and 200 ml of 0.1×SSC for 15 min each.
24. Dehydrate in a set of different concentrations (50%, 70%, 100%) of ethanol for 1 min each.
25. Dry the slices.
26. Soak the slides in gelatin solution for 1 min and air dry.
Radiographic autoradiography
See protocol 27.3 for details.