Primary adherent cell Giemsa staining

Primary adherent cell Giemsa staining

Dye preparation:

1. Weigh 0.5g of Giemsa powder and 22ml of glycerin;

2. Take a small amount of glycerin and mix well with Jimsa powder in a mortar and grind until no particles;

3. Pour the remaining glycerin into the mortar and incubate at 56 ° C for 2 h;

4. Add 33ml of pure methanol to mix, which is the Jimsa mother liquor, and store it in the brown reagent bottle;

5. Take 9 parts of Sorensen buffer with pH 6.80-7.30 and 1 part of Jimsa mother liquor to obtain the staining solution;

Adherent cell staining steps:

1. removing the culture solution in the culture vessel, and repeatedly rinsing the monolayer culture twice with the balanced salt solution;

2. Add a balanced salt solution, add an equal volume of methanol dropwise, while mixing, and let stand for 10 min;

3. Discard the 50% methanol-BSS mixture, add the fresh methanol solution to soak the cells for 10 min, then rinse the cells once with methanol;

4. Store the cells in the bottle after drying or directly stain;

5. Gemsa dye solution was added dropwise at a ratio of 2 ml/cm 2 , covering the cell layer and staining for 2 min;

6. Remove the dye solution, rinse off the pink background with tap water, then rinse the cells with deionized water;

7. Observe the single layer of moist cells with a microscope. If the cells are dry, re-make the cells and observe them again;

result:

The nucleus is stained purple or purplish blue, while the cytoplasm is stained light red;

Giemsa staining notes:

1. The dyeing solution should be used now, and the dyeing effect of the old dyeing solution is not good. The dyeing time should not exceed 48h;

2. Giemsa is extremely sensitive to pH, therefore, the pH of the buffer should be accurate, otherwise the dyeing effect is not good. When dyeing with a dyeing cylinder, as the dyeing time is prolonged, an oxide film (shiny) is often formed on the surface of the dyeing liquid to adhere to the glass sheet to form a stain, which is difficult to remove. Therefore, when dyeing with a dyeing cylinder, especially if it is not used, the oxide layer of the liquid surface should be scraped off with a small piece of filter paper before dyeing. After dyeing, the specimen should be immersed in water to rinse the dye solution;