Shanghai in situ hybridization experiment

(In Situ Hybridization) The nucleic acid of the labeled nucleic acid probe and cells or tissue Hybridization is called in situ hybridization. In situ hybridization technology, gene expression of a polypeptide or protein can be synthesized in situ. This method has high sensitivity and specificity, which can further explore the functional expression of cells and its regulatory mechanism from molecular levels. It has become an important means of today's cell biology and molecular biology research.

Experimental steps: Preparation of the pre-eulduct.

In situ hybridization * day for re-hydration and fixation, precisor, hybridization.

In situ hybridization for the second day, 1 is recovered by the probe, placed in -20c preservation (usually the probe can be reused for about ten times) 2 Add 50% formamide / 2XSSCT solution 1 ml, 60 ° C, 30 minutes, repeated once. 3 Replace 2XSSCT1ML, 60 ° C, stand for 15 minutes. 4 Replace 0.2xssct1ml, 60 ° C, stand for 30 minutes, repeat once. 5 Wash twice with MABT, five minutes each time, put it in the shaker gently shake. 6 Add 1 ml 1: 2: 7 solution at room temperature, time is one hour. 7 Press 1: 3000 to add enzymes to the enzyme in 1: 2: 7 solution, 4C refrigerator overnight.

In situ hybridization, 1) was used to replace the antibody solution with 1 ml of 10% heat-insumed MABT solution, and placed in a shaker for 25 minutes, then replace it with 1 mlmabt, 25 minutes. After replacement with 1 mlmabt solution, replace it with 1 mlmAbt solution for more than an hour, 25 minutes. 2) Wash three minutes with 1 ml 1 mm left-handed rice, for five minutes. 3) Transfer the embryo into the hex plate, sucks Staining Buffer, plus 300 ul BM Purple AP Substrate (substrate, with 5mm left-rotaryzole), and the tin foil paper outside of the 16-hole plate is taken away. Avoid shaking and color at room temperature. 4) Observe whether the embryo is starting to develop color 5) The substrate in the developing embryo is taken out, and 4% polymethylene formaldehyde is fixed after two or three times with PBST. 6) 4C refrigerator preserved.

Service content: 1 Cell specific mRNA transcriptional positioning, can be used for genetic spectrum, gene expression and genome evolution;

[123 ]

u0026 nBSP; 2 Infection tissue detection and positioning of viral DNA / RNA, such as EB virus mRNA, human papillomavirus and giant virus DNA detection;123]

Changes; 4 gene in chromosome;

5 Detection of chromosome changes, such as chromosome abnormalities and chromosome, etc.;

6 splitting interval cell genetics research, such as genetic disease prenatal diagnosis and certain genetic gene carriersDetermine, some tumors diagnosis and biological dose determination.