JRD213 in situ hybridization experiment

In situ hybridization experiment:
In situ hybridization can conduct a single cell study in a complex tissue of ingredients and is not affected by other components in the same tissue, Those cells are less and more convenient in intracellular DNA or RNA research in other tissues; while the in situ hybridization does not need to extract nucleic acids from tissues, there is a very high sensitivity for a target sequence in the tissue. The morphology of tissue and cells can be fully maintained, and the interrelation and functional state of tissue cells can be more accurately reflected.

In situ hybridization experimental reaction (as an example of DIG-cDNA probe):
1, the slice is routinely deprived to water, before dewaxing, placing tissue sections in a 60 ° C incubator bake 60min;
2,30% H2O2 1 part + distilled water 10 parts mix, 10min is 10 minutes at room temperature to inactivate endogenase, distilled water;
3, exposed mRNA nucleic acid fragment, slice, 3% lemon Sour fresh diluted gastric protease, 37 ° C for 30 min, PBS wash 5min x3, wash 1 time;
4, predecessor, wet box preparation u0026 mdash; u0026 mdash; dry hybrid box at the bottom of the hybrid box to maintain humidity. Press each 20 uL to add liquid. The thermostat is 40 ° C for 2 h, absorb excess liquid;
5, hybridization, press each slice 20 ul probe hybridization, add it on the slice. Cover the coverslide on the slice, the wax cover, the thermostat 42 ° C,;
6, after hybridization, remove the wax film, uncover the slide, 37 ° C 2 xiSSC wash 5 min X2; 37 ° C 0.5 xssc 15 min X1; 37 ° C 0.2 xssc wash 15min x1;
7, dropped closure, 37 ° C for 30 min;
8, dripped biotin-resistant mouse anti-ground, Xin, 37 ° C for 60 min, PBS wash 5 min X4;
9, 0.5 min X3 was washed at 37 ° C, PBS was washed 5 min X3;
10, a biotinized peroxidase, 37 ° C for 30 min, PBS was washed 5 min X4;
11, DAB Color, using the DAB color kit, 4 minutes of color, sufficient water after color;
12, Sukin reacted, water is washed back;
13, alcohol dehydration, xylene transparent, neutral gum
14, observed under the microscope.

Customers provide: 1. Tissue sample materials should be fresh and should be put into fixed liquid immediately after tissue (10% neutral formaldehyde, 4%) Multi-formaldehyde, in 2. Cell samples were centrifuged to discard the supernatant to add 3% glutaraldehyde fixation;
3. Paraffin slices, frozen slices, and cell climbs, slices, etc., please follow TemperatureAsk for transportation, store.

Submitted Products and Information:

2. Each slice provides a photo; 3. Experimental Report
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