In situ hybridization

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In situ hybridization

Service Description:

In situ Hybridization Histochemistry (ISHH), commonly known as In Situ Hybridization, is a molecular biology technique used to detect specific nucleic acid sequences within cells or tissues. This method involves using labeled DNA or RNA probes that bind to complementary sequences in the target cells. The process allows for the visualization of gene expression patterns directly in the tissue, preserving cellular architecture and providing high spatial resolution.

In situ hybridization can be categorized into three types based on the probe and target: DNA-DNA, DNA-RNA, and RNA-RNA hybridization. Additionally, it can be divided into direct and indirect methods depending on whether the probe is directly detected or requires an additional detection system.

The direct method uses radioisotopes, fluorescence, or enzyme-labeled probes that are visualized through autoradiography, fluorescence microscopy, or colorimetric reactions. The indirect method relies on hapten-labeled probes, which are then detected via immunohistochemical techniques, allowing for more sensitive and flexible detection strategies.

This technique offers several advantages, including the ability to study individual cells within complex tissues without interference from other cell types. It is especially useful for analyzing rare or scattered cells. Since it does not require the extraction of nucleic acids, it is highly sensitive to low levels of target sequences and preserves the morphological integrity of the tissue, enabling accurate analysis of cellular interactions and functional states.

Service Process:

1. Arrange the experiment based on the sample provided by the customer (fixed or embedded) and the type of sample.

2. After embedding or sectioning, design a specific probe according to the customer's requirements and perform a series of hybridization reactions.

3. (Optional) Film the resulting sections using a fluorescence microscope or confocal microscope, or an ordinary light microscope.

4. (Optional) Analyze the prepared slides, select a good area, and examine three different fields of view.

5. Provide a comprehensive experimental report, including detailed methods and figures showing the results of in situ hybridization.

Steps:

Material:

• Incubate in 0.1 M PBS (pH 7.2) for 5–10 minutes.

• Rinse in 0.1 M glycine/0.1 M PBS for 5 minutes.

• Treat with 0.3% Triton X-100/0.1 M PBS for 10–15 minutes.

• Wash with 0.1 M PBS for 5 minutes × 3 times, then incubate in proteinase K (1 μg/ml) at 37°C for 30 minutes.

• Fix in 4% paraformaldehyde for 5 minutes.

• Wash with 0.1 M PBS for 5 minutes × 2 times, then immerse in freshly prepared 0.25% acetic anhydride/0.1 M triethanolamine for 10 minutes.

Pre-hybridization: Add pre-hybridization solution dropwise at 42°C for 30 minutes.

Hybridization: Remove pre-hybridization solution, add 10–20 μl of hybridization solution (probe denatured and diluted in pre-hybridization buffer, 0.5 ng/μl), cover with a coverslip or wax film, and incubate overnight at 42°C.

Washing: Perform sequential washes in 4× SSC, 2× SSC, 1× SSC, 0.5× SSC at 37°C for 20 minutes; 0.2× SSC at 37°C for 10 minutes; 0.2× SSC and 0.1 M PBS for 10 minutes; and 0.05 M PBS for 5 minutes × 2 times.

• Block with 3% BSA/0.05 M PBS at 37°C for 30 minutes.

• Incubate with anti-alkaline phosphatase conjugate (1:5000 dilution) overnight at 4°C.

• Wash with 0.05 M PBS for 15 minutes × 4 times; TSM for 110 minutes × 2 times; fresh TSM for 210 minutes × 2 times.

Color development: Apply appropriate staining solution and incubate overnight at 4°C.

• Rinse in TE for 10–30 minutes to stop the reaction.

• Dehydrate with alcohol gradient and clear with xylene.

• Seal with neutral gum and observe under a microscope.