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DIG (Digoxigenin) labeled RNA probe In situ hybridization protocol
实验步骤
1. Deparaffinization
If using formaldehyde fixed paraffin embedded sections. For frozen sections, please start at section 2
Before proceeding with the staining protocol, the slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section.
1) Xylene
2) 100% ethanol
3) 95% ethanol
Place the slides in a rack, and perform the following washes:
4) Xylene: 2 x 3 minutes
5) Xylene 1:1 with 100% ethanol: 3 minutes
6) 100% ethanol: 2 x 3 minutes
7) 95% ethanol: 3 minutes
8) 70 % ethanol: 3 minutes
9) 50 % ethanol: 3 minutes
10) Running cold tap water to rinse
Keep the slides in the tap water until ready to perform antigen retrieval. At no time from this point onwards should the slides be allowed to dry. Drying out will cause non-specific antibody binding and therefore high background staining.
2. Antigen retrieval
Digest with 20 µg/ml proteinase K in pre-warmed 50 mM Tris for 10 to 20 minutes 37°C. The time of incubation and concentration of proteinase K may require some optimization.
| The concentration of proteinase K and the incubation time for this step will require optimization. We can recommend trying a proteinase K titration experiment to determine the optimal conditions. Insufficient digestion will result in a reduced hybridization signal. Over digestion will result in poor tissue morphology, making localization of the hybridization signal very difficult. The concentration of proteinase K needed will vary depending upon the tissue type, length of fixation, and size of tissue. |
3. Rinse slides five times in distilled water
4. Immerse slides in ice cold 20% (v/v) acetic acid for 20 seconds. This will permeabilize the cells to allow access to the probe and the antibody.
5. Dehydrate the sections by washing for approximately one minutes each wash in 70% EtOH, 95% EtOH and 100% EtOH then air dry.
6. Add 100 µl hydridization solution to each section
| Reagent | Final conc | Amount to use per 1 ml of solution |
| Formamide | 50% | 500 µl |
| Salts | 5x | 250 µl |
| Denhardt’s solution | 5x | 100 µl |
| Dextran sulphate | 10% | 200 µl |
| Heparin | 20 U / ml | 10 µl |
| SDS | 0.1% | 10 µl |
Salt solution:
4 M NaCl 100 mM EDTA 200 mM Tris-HCl pH 7.5 100 mM NaH2 PO4 .2H2 O 100 mM NaH2 PO4
Denhardt’s solution(100x):
10 g Ficoll 10 g PVD (polyvinylpyrrolidine) 10 g BSA (Bovine Serum Albumin) 500 ml sterile dH2 O
7. Incubate the slides 1 hour in hybridization chamber at the desired hybridization temperature. Typical hybridization temperatures range between 55 and 62°C (for more details, see notes from section 9 below)
8. Dilute the probes in hybridization solution ready in PCR tubes. Heat for 95°C for 2 minutes on a PCR block. This will dehybridize the RNA or DNA probe. Chill on ice immediately to prevent rehybridization.
9. Drain off the hybridization solution. Add 50 to 100 ul of diluted probe per section (ensure the entire sample is covered). Incubate in the hybridization chamber at 65°C overnight. Whilst incubating, the sample on the slide can be covered with a cover slip to prevent evaporation.
During this step, the RNA probe will hybridize to the corresponding mRNA, or the DNA probe will hybridize to the corresponding cellular DNA.
| The hybridization temperature will require optimization depending on the sequence of the probe used, as well as the cell / tissue type. This temperature should be optimized for each tissue type analyzed. Hybridization temperatures used range from 55 to 62°C. The optimal hybridization temperature for the probe depends on the percentage of bases present in the target sequence. The concentration of cytosine and guanine in the sequence are an important factor. |
10. Stringency washes:
Solution parameters such as temperature, salt and/or detergent concentration can be manipulated to remove any non-identical interactions (i.e. only exact sequence matches will remain bound).
To prepare 1 liter of 20 x SSC: For 1 liter:
175.3 g NaCl (3 M) 88.2 g Na citrate 800 ml sterile dH2 O Adjust to pH 5 using Citric acid, top up to 1 litre and then autoclave.
Wash 1 50% formamide / 2 x SSC 3 x for 5 min, 37-45°C.
To wash away any excess probe and the hybridization buffer. Higher temperatures (up to 65°C) can be used for short periods of time, but this can wash off too much of the hybridized probe RNA / DNA if left for too long.
Wash 2 0.1-2 x SSC 3 x for 5 minutes, 25°C to 75°C.
This step removes non-specific and/or repetitive DNA / RNA hybridization. The less concentrated the salt solution and the longer the duration of the wash and the temperature, the higher the stringency and the more DNA / RNA will be removed.
| Optimization of temperatures for stringency washes can be difficult to work out, but the following guidelines can help: Very short DNA/RNA probes (0.5-3 kb) or very complex probes, the washing temperature should be lower (up to 45°C) and the stringency lower (1x-2 x SSC). Single-locus or large probes, the temperature should be around 65°C and the stringency high (below 0.5 x SSC). The temperature and stringency should be highest for repetitive probes (such as alpha-satellite repeats). |
11. Wash twice in MABT (maleic acid buffer containing Tween 20) for 30 minutes at room temperature.
MABT is gentler than PBS and is more suitable for nucleic acid detection.
12. Dry the slides
13. Transfer to a humidified chamber and add 200 µl blocking buffer to each section (MABT 2% BSA, milk or serum). Block for one to two hours, room temperature.
14. Drain off the blocking buffer. Add the anti-‘label’ antibody at the required dilution in blocking buffer. Check the antibody datasheet for a recommended concentration. Incubate for one to two hours at room temperature.
15. Wash slides 5 times with MABT, 10 minutes for each wash, room temperature
16. Wash the slides 2 x for 10 minutes room temperature with prestaining buffer (100 mM Tris pH 9.5, 100 mM NaCl, 10 mM MgCl2 ).
17. Fluorescence – please proceed to step 18 Other – return slides to humidity chamber and follow manufacturer’s instructions for color development.
18. Rinse slides in distilled water.
19. Air dry the slides for around 30 minutes. Wash in 100% ethanol, then air dry thoroughly.
20. Mount using DePeX mounting solution.
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