Acridine orange fluorescent staining of primary cell DNA and RNA
Acridine orange (AO) is a commonly used fluorescent dye. Acridine orange has affinity with DNA and RNA in primary cells, but has certain specificity, that is, fluorescence of different colors after binding, DNA is Bright green, while RNA is orange to red. This method is simple and quick, and can both stain primary cells and stain primary cells. It can be used for direct staining to observe live primary cells or for fixed staining to observe primary cells. Acridine orange is less toxic to live primary cells, and can be used to directly stain live primary cells and perform fluorescence microscopy with 1Ã—10 -4 -2Ã—10 -4 , but it is not durable, and it is better to use fixed staining. .
1. Covering the monolayer to culture primary cells;
2. Fixing agent: 95% ethanol or acetic acid / methanol (currently available);
3. Acridine orange: Prepare 1% dry solution with normal saline. When used, it is diluted with PBS to a staining solution of 1Ã—10 -4 - 2 Ã— 10 -4 mol / L pH 4.5 - 5.0;
4. 0.1 mol/L CaCl 2 ;
5. 1% acetic acid;
1. Remove the cover plate monolayer culture primary cells from the bottle, and immediately rinse with warm Hanks solution for 3-5 seconds to remove serum from the culture medium;
2. Put in 95% ethanol or acetic acid/methanol for 15-30 minutes, touch the edge of the cover sheet with filter paper, and cover the plate slightly dry;
3. Acidify with 1% acetic acid for 30 s;
4. Stain with 1 Ã— 10 -4 - 2 Ã— 10 -4 acridine orange dye solution for 30 s or 1 min;
5. Treatment with 0.1 mol / L CaCl 2 for 30 s - 2 min;
6. Rinse PBS 3 times, every few seconds;
7. PBS seal, directly observe and photograph under the fluorescence microscope;
8. Note: PBS should be added at any time during the observation to avoid dryness of the specimen. If it is observed with an oil-immersed mirror, it should be covered with a large cover slip on the original cell cover sheet to ensure that the primary cells face up and are observed with a non-fluorescent oil immersion mirror. Primary cell cover specimens can be observed in 95% ethanol for several days, such as fading, and then stained with AO. Under the microscope, the DNA was emerald green and the RNA was red.
The primary nucleus contains green DNA, and the cytoplasm and nucleolus are red in color.
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