In situ hybridization
In situ hybridization Service Description: In situ Hybridization Histochemistry (ISHH), commonly known as In Situ Hybridization, is a molecular biology technique that falls under the category of solid-phase hybridization. It uses labeled DNA or RNA probes to detect specific nucleic acid sequences within cells in their original tissue environment. This method allows for precise identification and localization of genetic material at the cellular level. There are three main types of in situ hybridization based on the probe and target nucleic acid: DNA-DNA hybridization, DNA-RNA hybridization, and RNA-RNA hybridization. Additionally, it can be categorized into direct and indirect methods depending on whether the probe's label is directly detected. The direct method typically involves radioisotopes, fluorescent dyes, or enzyme-labeled probes, which are visualized using autoradiography, fluorescence microscopy, or colorimetric assays. The indirect method employs hapten-labeled probes, which are then detected via immunohistochemical techniques to visualize the hybridized complex. In situ hybridization offers significant advantages, especially when studying rare or scattered cells within complex tissues. It avoids the need for isolating nucleic acids from the tissue, making it highly sensitive even for low-abundance target sequences. Moreover, it preserves the structural integrity of the tissue, enabling more accurate analysis of cell interactions and functional states. Service Process: 1. We arrange the experiment based on the sample provided by the customer, whether it is fixed or embedded, and the type of sample. 2. After embedding or sectioning, we design a specific probe according to the customer’s requirements and proceed with a series of reactions. 3. (Optional) We can film the high-quality sections and analyze them under a fluorescence microscope or a confocal microscope. 4. (Optional) We analyze the prepared slides, select the best area for observation, and examine three different fields of view. 5. We provide a comprehensive report that includes detailed experimental procedures and relevant images of the in situ hybridization results. Steps: Material: • Soak the tissue in 0.1 M PBS (pH 7.2) for 5–10 minutes. • Incubate 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. Add proteinase K (1 μg/ml) and incubate 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: Apply pre-hybridization solution dropwise at 42°C for 30 minutes. Hybridization: Remove the pre-hybridization solution, add 10–20 µl of hybridization solution (probe denatured and diluted in pre-hybridization solution, 0.5 ng/µl), cover with a coverslip or wax film, and incubate overnight at 42°C. (Negative control included.) Washing: Perform a series of 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-anti-serum alkaline phosphatase complex (diluted 1:5000 in antibody diluent) 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: Add appropriate coloring solution to the slide and let it develop overnight at 4°C. • Rinse the slide in TE for 10–30 minutes to stop the reaction. • Dehydrate through alcohol gradients and clear with xylene. • Mount with neutral gum and observe under a microscope. Dongguan Pinji Electronic Technology Limited , https://www.iquaxusb4cable.com