Giemsa staining experiment

Summary

Staining of cells allows (1) coloring of cells for easy observation and (2) detection of apoptotic cells.

Operation method

dyeing experiment

Principle

The Giemsa stain consists of azure, eosin. The staining principle and results are basically the same as Ritter's stain. Eosinophilic particles are basic proteins that bind to the acidic dye eosin and stain pink, called eosinophilic substances; nucleolar proteins and lymphocyte cytoplasm are acidic and bind to the basic dyes methylene blue or azure, staining purplish-blue, called basophilic substances; neutral particles are in an equipotential state that binds to eosin and methylene blue, staining mauve, called neutral substances.PH has some effects on cell staining. Various components of the cell are not proteins, because proteins are amphoteric electrolytes, the charge with the solution PH, in a more acidic environment, the positive charge increases, easy to combine with eosin, stained red; in a more alkaline environment, the negative charge increases, easy to combine with the blue or azure, stained bluish.

Materials and Instruments

In situ hybridization slides
Giemsa staining solution Sodium phosphate buffer Ethanol Xylene
Staining trays Incubator Filter paper

Move

1. Place the developed slide on a slide holder and immerse it in a staining dish containing water.

(1) 1 tray: pH 6.8 10 mmol/l sodium phosphate buffer, 25-fold dilution of the prepared Giemsa stain.

(2) 3 trays: water.

(3) Dehydration series solution:

(1) 3 trays: 50%, 70% and 95% ethanol.

(② 2 trays: holding 100% ethanol.

③ 3 trays: containing xylene.3. Stain the slide in 25-fold dilution of Giemsa's staining solution for 20 s (depending on the staining time to determine the strength of the staining), and immerse the slide in water three times for 2 min each time.

4. The slides were continuously treated with a series of ethanol dehydrating solutions, soaking for 2 min in each dish, and then transferred to a xylene dish, soaking for 2 min each time, for a total of 3 times.5. In a fume hood, press the slides for fixation as follows: Using a pair of flat-tipped forceps (held in the left hand), remove a slide from the xylene and hold the abrasive end horizontally. Add 4 drops of Permount to the other end (where the slides will be), take a clean coverslip in the left hand and place it very slowly on the slide (do not allow the slide to dry out before adding the press-fixing medium and the coverslip, as tiny air bubbles can cause artifacts).6. Using 3MM filter paper, carefully pipette excess solid medium/xylene along the edge of the coverslip and wipe the back of the slide (do not wipe the coverslip).7. Place the slide flat on a cardboard tray and incubate at 42°C for 2 days to solidify.8. Carefully scrape the back of the slide with a razor blade to remove any residual latex, dye and fixative media. Wipe off the dust with lens paper or plain cotton paper. Place in a slide box and, if necessary, label the slide again.
9. The slide is viewed under a microscope, and the light can be adjusted according to the strength of the signal.

Caveat

1. Do not store slides in an upright position, so that the fixing medium has not yet solidified.

2. Cell staining is very sensitive to the concentration of hydrogen ions, staining with the warps must be clean, to be free of acid and alkali contamination.

3. Preparation of anguish solution must use high-quality methanol, dilution of staining must use buffer, rinse must use water should be nearly neutral, otherwise it can lead to a variety of cell staining reaction abnormality, so as to identify the difficulties, and even cause the wrong.

Common Problems

Staining experiments are related to the staining concentration, the number of cells, and the room temperature; a light staining solution, a low temperature, and more cells will result in a longer staining time, and vice versa, the time can be reduced, and the amount of staining solution can be increased or the time can be increased if necessary. Before rinsing, it is necessary to observe the staining under a low-power microscope to see whether the nucleus is distinct and whether the staining is clear.

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