In Situ Hybridization Requirements and Procedures

Summary

In situ hybridization can be used for (1) solid-phase molecular hybridization and (2) labeled DNA or RNA as probes to detect specific nucleic acid sequences in tissue cells in situ.

Operation method

In Situ Hybridization Requirements and Procedures

Principle

In situ hybridization can be divided into direct and indirect methods according to whether the marker of the probe is directly detected or not. The direct method mainly uses radioisotope, fluorescence and certain enzyme-labeled probes to hybridize with the target nucleic acid, and the hybridization is directly displayed by radioautography, fluorescence microscopy or color-forming enzymatic reaction, respectively. Indirect methods generally use semi-antigens to label the probes, and finally, through immunohistochemistry to localize the semi-antigens, indirectly display the hybrids formed between the probes and the target nucleic acids.

Materials and Instruments

Fresh tissue or cell samples
PBS PB Glycine paraformaldehyde Denhardt's solution Antibody diluent Pre-hybridization solution Color development solution Anhydrous ethanol
Water bath Centrifuge Nylon membrane Spotter

Move

(i) Sampling and frozen section:

The animal is anesthetized with 3% pentobarbital sodium, the chest cavity is opened, the heart is exposed, the right auricle is punctured, and the tip of the needle is punctured into the left ventricle to perfuse it with saline (the perfusion volume is approximately twice the animal's body weight), and an equal amount of 4% paraformaldehyde is injected. (See Figure 2-7). The material is taken and placed in 4% paraformaldehyde and post-fixed for 4 hr. It is rinsed 4-5 times with 0.1 M PBS immersion (fluid change: 1 time/hr). The tissue was blocked into 30% sucrose/0.1M PBS solution (4°C) and frozen sectioned after 1-2 days. The sections were mounted on special slides for in situ hybridization with a section thickness of 15-20μm.

(ii) Probe preparation and detection (quantitative):

1. Random primer preparation of cDNA nucleic acid probe Take DIG DNA labeling detection kit as an example:

(1) Probe preparation:

① Template DNA (0.5-3 μg) 15 μl, denature at 100℃ for 10 min, ice bath for 5 min.

② Add Hexanucleotide mix 2 μl, dNTPmix 2 μl, Klenow Enzyme 1 μl into the ice bath, the total volume of the reaction is 20 μl. Incubate the reaction overnight at 37°C. ③ Add 20 μl of the above 20 μl of Hexanucleotide mix into the ice bath.

③ Add 4 M LiCl 2.5 μl into the above 20 μl of labeling product, 75 μl (pre-cooled in anhydrous ethanol, mix gently, and place at -20℃ for 2 hr. Centrifuge at 4℃ for 12,000 g×15 min, and discard the supernatant. 70% ethanol (pre-cooled), 50 μl wash, and then 7,500 g×5 min, and then discard the supernatant, and then air-dry the precipitate, and then add 50 μl TE to dissolve the precipitation, and then store it at -20℃. Store at -20℃.

(2) Probe sensitivity test:

① Sample dilution: take 1 μl of dig-labeled probe and dilute it with ddH2O in 1:10, 1:100, 1:1000, 1:1000, 1:10000 gradient.

② Take a piece of nylon membrane similar to the size of the spotter, mark the direction, soak it in ddH2O for 1 min, soak it in 6×SSC for 10 min, place it on the spotter, and suction it under negative pressure for 5 min.

(iii) Spot the above samples on the nylon membrane, and continue suction for 10 min.

④Remove the nylon membrane, irradiate it at 10 cm under the UV lamp for 5 min, and let it dry.

⑤ Place the membrane in appropriate amount of prehybridization solution (5-10 ml) and prehybridize at 37℃ for 10 min.

(6) Add Anti-dig antibody (1:5000) and hybridize for 30 min at 37℃.

(vii) Wash the membrane: 2×SSC/0.1% SDS at room temperature for 10 min×2 times.

(8) Color development: 15 ml TSM2 with 300 μl of color development solution (NBT/BCIP), 37 ℃ avoiding light color development for 30 min.

2. PCR method to prepare cDNA nucleic acid probe:

(1) Probe preparation:

Take PCR DIG Probe Synthesis Kit as an example:

In a 0.5 ml centrifuge tube, sequentially add:

PCR Primer 1 (10 pM) 2 μl;

PCR Primer 1 (10 pM) 2 μl;

Plasmid DNA template (10-100 pg) 2 μl;

PCR DIG mix 2 μl (containing dNTP and DIG-11-dUTP);

dNTPmix 2 μl;

10×PCR buffer 5 μl;

Taq enzyme (2 u/μl) 1 μl;

Add appropriate amount of ddH2O to make a total volume of 50 μl.

① Centrifuge the above mixture, and immediately place it on the PCR instrument to perform amplification.

In general: pre-denaturation at 93 ℃ for 3-5 min, enter the amplification phase: 93 ℃ 45 s → 58 ℃ 45 s → 72 ℃ 60 s, cycle 30-35 times, and finally hold at 72 ℃ for 7 min.

② Add 4 M LiCl 12.5 μl, pre-cooled anhydrous ethanol 375 μl to the above PCR products, mix gently and place at -20 ℃ for 2 h, 12,000 g × 15 min, discard the supernatant. 70% ethanol (pre-cooled) 120 μl wash the precipitate, centrifugation 7,500 g × 5 min, discard the supernatant, air-drying the precipitate, add TE 50 μl to solubilize, and -20 ℃ to store for backup.

(2) Probe detection:

Perform agarose gel electrophoresis and observe the content of DIG-DNA probe under UV light (visual inspection method).

(C) In situ hybridization reaction (using DIG-cDNA probe as an example):

1. 0.1 M PBS (pH 7.2) immersion for 5-10 min.

2. 0.1 M glycine/0.1 M PBS immersion for 5 min. 3. 0.3% Triton (pH 7.2) immersion for 5-10 min.

3. 0.3% TritonX-100/0.1 M PBS for 10-15 min. 4. 0.1 M PBS for 5 min.

4. 0.1 M PBS washed for 5 min×3 times, proteinase K (1 μg/ml) was added, and incubated at 37℃ for 30 min.

5. 4% paraformaldehyde was immersed for 5 min.

6. 0.1 M PBS washed 5 min×2 times, immersed in freshly prepared solution containing 0.25% acetic anhydride/0.1 M triethanolamine for 10 min.

7. Pre-hybridization: add appropriate amount of pre-hybridization solution dropwise, 42°C for 30 min.

(1) Hybridization: Pour off the prehybridization solution, add 10-20 μl of hybridization solution (0.5 ng/μl of probe diluted in prehybridization solution after denaturation) to each slide, cover with a coverslip or wax film, and incubate overnight at 42℃.

(2) Washes:

4×SSC, 2×SSC, 1×SSC, and 0.5×SSC were each washed for 20 min at 37℃;

0.2×SSC washed at 37℃ for 10 min; 0.2×SSC and 0.1 M PBS were half-washed for 10 min each;

0.05 M PBS washed 5 min × 2 times.

10. 3% BSA/0.05 M PBS was encapsulated at 37℃ for 30 min.

11. Dropwise addition of anti-digoxin-anti-serum alkaline phosphatase complex (diluted 1:5000 with antibody diluent) Incubate at 4℃ overnight.

12. Wash with 0.05M PBS for 15 min×4 times; TSM1 for 10 min×2 times; freshly prepared TSM2 for 10 min×2 times.

13. Color development: Appropriate amount of color development solution was added dropwise on the slide and incubated at 4°C overnight away from light.

14. Place the slide in TE for 10-30 min to terminate the reaction. Alcohol gradient dehydration, xylene degreasing, neutral gum sealing. 15.

15. Observe the results under microscope.

Caveat

Hybridization solution:In addition to a certain concentration of labeled probe, the hybridization solution also contains higher concentrations of salts, formamide, dextran sulfate, bovine serum albumin and carrier DNA or RNA.Higher concentrations of Na+ in the hybridization solution can increase the hybridization rate and can reduce the electrostatic binding between the probe and the tissue specimen. Formamide can make Tm lower, hybridization solution containing every 1% of formamide can make the hybridization temperature of RNA:RNA, RNA:DNA, DNA:DNA lower by 0.35℃, 0.5℃ and 0.65℃, respectively.Therefore, the addition of an appropriate amount of formamide to the hybridization solution can avoid the destruction of tissue morphology and structure and the detachment of the specimen caused by the high hybridization temperature. Dextran sulfate can combine with water, thus reducing the effective volume of the hybridization solution and increasing the effective concentration of the probe to achieve the purpose of improving the hybridization rate (especially for double-stranded nucleic acid probes).The addition of bovine serum albumin and carrier DNA or RNA, etc. to the hybridization solution is all about blocking the non-specific binding between the probe and the structural components of the tissue in order to reduce the background.Probe concentration:Probe concentrations vary slightly depending on the type and experimental requirements, generally 0.5--5.0 μg/ml (0.5--5.0 ng/μl). The optimum probe concentration can only be determined experimentally.Probe length:Generally it should be between 50-300 bases, and the longest length should not exceed 400 bases. Short probe is easy to enter the cell, high hybridization rate and short hybridization time.

Common Problems

1. Tissue sampling: Tissue sampling should be as fresh as possible. Because of the rapid degradation of tissue RNA, fresh tissue and cultured cells should be fixed within 30 min. 2.

2. The purpose of fixation is:

(1) Maintain the cell structure;

(2) Maximize the level of intracellular DNA or RNA;

(3) Make the probe easy to enter the cell or tissue.

The most commonly used fixative is paraformaldehyde, which, unlike other aldehyde fixatives (e.g. glutaraldehyde), does not produce extensive cross-linkages with proteins, and thus does not interfere with probe penetration into cells or tissues.

3. Enhance tissue permeability and nucleic acid probe penetration:

(1) Dilute acid treatment and acid anhydride treatment: To prevent electrostatic binding between the probe and basic proteins in the tissue to reduce the background, the specimen can be treated with 0.25% acetic anhydride for 10 min before hybridization. After treatment with acetic anhydride, the basic groups in the tissue proteins are blocked through acetylation. Tissue and cell specimens can also be treated with 0.2 M HCl for 10 min, dilute acid can denature the basic protein, combined with protease digestion, easy to remove the basic protein.

(2) Decontaminant treatment: the purpose of decontaminant treatment is to increase the permeability of the tissue to facilitate the entry of hybridization probes into the tissue cells, the most commonly used decontaminant is Triton X-100. note: excessive decontaminant treatment not only affects the morphology and structure of the tissue, but also causes the loss of target nucleic acids.

(3) Protease treatment: Protease digestion can expose the target nucleic acids that have been masked after fixation to increase the accessibility of the probe to the target nucleic acids. Commonly used proteases are proteinase K (proteinase K), as well as streptavidin (pronase) and pepsin.

4. Incubation in hybridization buffer:

Incubate with probe-free hybridization buffer for 2 hr at hybridization temperature before hybridization to block sites in slides and specimens that may bind non-specifically to the probe for the purpose of background reduction.

5. Prevention of contamination:

Since RNAase may be present on the skin of the fingers and on the glassware used in the laboratory, in order to prevent its contamination from affecting the results of the experiments, sterilized gloves should be worn throughout the entire pre-hybridization process, and the glassware and tweezers used in the experiments should be baked at high temperatures (180°C) one day prior to the experiments for the purpose of eliminating the RNAase. The solutions used before and during hybridization should be sterilized by autoclaving.

6. Denaturation of double-stranded DNA probes and target DNA:

Both the probe and the target nucleic acid must be single-stranded when the hybridization reaction is performed. If hybridization is performed with a double-stranded DNA probe (including when detecting RNA), the double-stranded DNA probe must be denatured prior to hybridization. The hybridization reaction must be carried out immediately after denaturation of the probe, otherwise the unstranded probe will be re-denatured again.

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