In situ hybridization technology-Shanghai Medicilon

In situ hybridization (insituhybridization) uses a specific labeled nucleic acid of known base sequence as a probe to specifically detect the nucleic acid in tissues and cells in accordance with the principle of complementary base pairing in situ to form a hybrid, which is then labeled with The corresponding monitoring system of the substance can locate the nucleic acid in the cell in situ by immunohistochemistry. In situ hybridization can be divided into intracellular and tissue section in situ hybridization according to the test substance; according to the probes and nucleic acid detection, it can be divided into DNA-DNA, RNA-DNA, RNA-RNA hybridization. According to different labeling methods, it can be divided into radioactive probes and non-radioactive probes.
The basic methods include: fixation, sectioning, hybridization, color development and other main steps.

(1) The material is fixed with the specimen

1. Material extraction: The material used for in situ hybridization should be as fresh as possible. In order to prevent the degradation of RNA, the material should be taken quickly, and the material should be fixed or frozen as soon as possible after the material is taken.

2. The tissues or cells that are fixed for in situ hybridization must be fixed. The purpose of fixation is to ① maintain the original structure of the cell morphology; ② maximize the preservation of the level of DNA or RNA in the cell; ③ make the probe easy to enter the cell or tissue .

3. Fixatives: Common fixatives include 10% formaldehyde, 4% paraformaldehyde, glacial acetic acid-alcohol mixed solution, Bouin’s fixative, etc. These fixatives have different advantages and disadvantages, so the best fixative should be selected according to the specific experimental object.

4. Fixing method 　 After taking tissue samples, quickly put them into fixative solution for 2 to 4 hours. The size of the sampled tissue is 1cm×1cm×0.5cm, which should not be too large. If necessary, the animal tissue can be perfused and fixed, and then the tissue is taken into 20% sucrose according to the requirements, overnight at 4 ℃, and the next day can be frozen and sliced.

(2) Preparation of sections and cell specimens

1. Slide processing 　Because in situ hybridization is generally performed on slides, slides must be kept clean and free of any nuclease contamination. Generally, the glass slide can be soaked in washing powder overnight, rinsed with running water, soaked in acid for 4-8 hours, then rinsed with running water, and washed with double distilled water 2 to 3 times. Bake in 160℃ oven for 2～4h. It can also be sterilized by autoclaving at 15 pounds for 20 minutes to eliminate the nuclease on the slide.

2. Tissue sectioning and pretreatment
   (1) Frozen section: fresh tissue can also be placed in liquid nitrogen for rapid quenching after taking the material. After freezing section, 4% paraformaldehyde is fixed for 5 to 10 minutes, and can also be stored at -70°C until use. Before hybridization, the slices were quickly returned to room temperature and dried, washed three times with 0.1mol PBS, washed with 2×SSC for 10 minutes, and added with pre-hybridization solution and incubated at room temperature for 1 hour.
   (2) Paraffin section: After the tissue is fixed, conventional paraffin embedded sections can be stored for a long time and can be used for in situ hybridization at any time. Before hybridization, slices were dewaxed 2 times with xylene for 10min each time; ②washed with pure alcohol 2 times with 5min each time; ③air dried for 5min; ④ gradient alcohol rewashed 95%, 80%, 70% for 1min each; ⑤0. Wash 3 times with 1 mol PBS for 5 minutes each time; ⑥ 2×SSC for 10 minutes; ⑦ add prehybridization solution dropwise and incubate for 1 hour at room temperature in a wet box.
   (3) Cultured cells: add 200 μl of a cell suspension with a concentration of 1×105/m1 to each slide, and air-dry for 1 to 2 minutes to prepare a cell smear, or directly use a cultured cell cover slip. The above cell pieces were fixed with alcohol or paraformaldehyde for 5 minutes, and the remaining steps were the same as frozen sections.

(3) Reagent preparation
Gloves are required during the preparation of the solution. The liquid preparation uses ultra-clean water. The bottles used are baked at 160 ℃ for 4 hours. The main purpose is to remove RNase.

1. DEPC water 　 Add DEPC to ultrapure water at a concentration of 1‰, mix thoroughly and let stand overnight, autoclave for 15~20min, then store at room temperature and avoid dust.

2. 0.1mol PBS　pH 7.4
   Liquid A: 0.1mol NaH2PO4 is 1.56g of NaH2PO4 (MW: 156.01, 2H2O); DEPC water is 100.00m1.
   Liquid B: 0.1 mol Na2HP04 is Na2HPO4 (MW: 358.14, 12H2O) 17.907g; DEPC water 500.00m1.
   Liquid A: Liquid B = 9.5:40.5 Add NaCl to make its concentration reach 0.85%.

3. 20×SSC　 is NaCl (3mol/L) 8.765g; Na3C6H50H2O7.2 (0.3mol/L) 4.410g; DEPC water 50.00m1.

4. 4% paraformaldehyde (pH 7.4) is paraformaldehyde 4g; 0.1mol PBS 100ml.

5. Buffer
   (1) Buffer I (pH 7.5) is 1mol Tris-HCl 100ml; 5mol NaCl 20ml; disinfection ultra-clean water is added to 1000ml.
   (2) Buffer II (pH 9.5) is 1mol Tris-HCl 100ml; 5mol NaCl 20ml; 1mol MgCI 50ml; disinfection ultra-clean water is added to 1000ml.
   (3) Buffer III (pH 9.5) is 1mol Tris-HCl 100ml; 1mol EDTA 5ml; 5mol NaCl 20ml; 1mol MgCI 50ml; disinfection ultra-clean water is added to 1000ml.

6. Deionized formamide　Add 5g each of anion and cation exchange resin to 50ml formamide, and use supernatant after the resin sinks.

7. 50% Dextran Sulfate 　 5g Dextran Sulfate plus 10ml DEPC water, keep stirring to completely dissolve.

8. 50×Denhardt’s solution is 1% Fico11 type 400 (polysucrose) 0.2g; 1% PVP (polyvinylpyrrolidone) 0.2g; 1% BSA (bovine serum albumin) 0.2g; DEPC water 20.0ml.

9. Pre-hybridization solution is deionized formamide 5.0m1; 20×SSC 2.0m1; salmon sperm DNA (10mg/m1); 0.5ml (boiling water denaturation for 10min); yeast tRNA (10mg/m1) 0.25m1; 50% dextran sulfate 2.00m1; 50×Denhardt? Tribulus solution 0.20ml.

(4) Hybridization and color development

1. Hybridization Add the labeled probe to the pre-hybridization solution to become the hybridization solution (probe concentration is 1μg/m1). After the section is briefly immersed in 2×SSC, the hybridization solution is added dropwise and incubated in the built-in wet box at 37℃ or 42℃ overnight. Then, it was immersed in 2×SSC at room temperature for 1 h, l×SSC at room temperature for 1 h, 0.5×SSC at 37°C for 30 min, and 0.5×SSC at room temperature for 30 min. Be careful not to dry the sections when adding the hybridization solution.

2. Color development 　 The following steps are carried out at room temperature.
   (1) Buffer I was washed for 1 min.
   (2) Incubate the sections with buffer I containing 2% normal sheep serum and 0.3% TritonX-100 for 30 min.
   (3) Dilute goat anti-Dig antibody with buffer I containing 1% normal sheep serum and 0.3% TronX-100.
   (4) Drop the antibody dilution onto the slices, cover with parafilm, and incubate at 4°C in a wet box overnight.
   (5) Wash with buffer I for 10 minutes.
   (6) Buffer II was washed for 10 minutes.
   (7) Add color developing solution, cover with parafilm, incubate at room temperature for 2 to 20 hours in the dark, and examine at any time. When the color development is satisfactory, add buffer III to stop the color development.
   The coloring solution is prepared immediately before use, and its formula is: ① NBT 45μl; ② X-Phospllate solution 35μl; ③ Levamisole 2.4mg; ④ Buffer II is added to 10ml.
   (8) Conventional dehydration, transparency and sealing.
   Results: The positive hybridization signal was purple-blue particles.

(5) In situ hybridization control test
As with the immunohistochemical staining control, in order to prove the accuracy of the in situ hybridization experiment operation and the specificity of the experimental results, a series of control experiments need to be set up. For the setting and significance of various control experiments, please refer to the in situ hybridization monograph. Only a few commonly used control experiments are introduced below.

1. Eliminate the labeling probe. When doing in situ hybridization, replace the hybridization solution with PBS or prehybridization solution. The result should be negative. This is a simple and meaningful negative control experiment. If you omit the labeled probe and still have a positive reaction, this must be a false positive.

2. Self-control: Use other structures on the same tissue section or smear that are not related to the target sequence as controls. The positive and negative structures are in the same field of view, confirming each other, which itself is a specific control for positive reactions.

3. Negative control 　 hybridize with tissue samples known not to have the corresponding target sequence, the result should be negative. The negative control can exclude false positive results caused by non-specific hybridization reactions and other factors during the hybridization process.

4. Positive control 　Use tissue sections with known target sequences to be treated in the same way as the specimens to be tested. The positive control can prove that the steps in the hybridization process and the reagents used are in line with the standard, and the experimental method is reliable. Especially when the specimen to be tested is negative, the positive reaction to the photo can rule out the possibility of false negative of the specimen to be tested. Therefore, if the hybridization result is expected to be negative, a positive control must be set. When the positive control does not develop color, it proves that there is a problem in the hybridization process or the reagent used.