Ion cytochemistry can be used to show the distribution of intracellular ion localization. Currently, it is more commonly used to show the distribution of intracellular calcium, usually in conjunction with EDX energy spectroscopy. Intracellular calcium ion distribution is highly compartmentalized, forming calcium pools with different concentrations of calcium ions. Under normal conditions, intracellular calcium is distributed in the cytoplasm, mitochondria, nucleus and other parts of the cell; under most pathological conditions (e.g., ischemia, hypoxia, intoxication, etc.), intracellular calcium can be elevated, and a lot of the calcium that enters the cell enters the mitochondria. Intracellular calcium ion cytochemistry is based on the principle that phosphate or oxalate is first used to react with calcium ions to form a precipitate in situ, and then pyroantimonate is used to react with calcium pyroantimonate, replacing the phosphate or oxalate to form calcium pyroantimonate, which is electron-dense under an electron microscope.

Operation method

Calcium ion cytochemistry specific methods

Materials and Instruments

Tissue
Potassium phosphate Glutaraldehyde Sucrose Potassium pyroantimonate Osmuric acid

Move

1. The tissue is cut into small pieces of about 1 mm³ and fixed with 0.09 mol/L potassium phosphate (or potassium oxalate)-3% glutaraldehyde (pH 7.3, adjust the pH with 0.1%-1% KOH) for more than 4 h at 4℃. Microwave irradiation is also recommended at the early stage of fixation to accelerate the penetration of the fixative into the cells (for the use of microwave irradiation, see the relevant section). 2.

2. After fixation, rinse the cells with 0.09 mol/L potassium phosphate (or potassium oxalate)-4% sucrose (pH 7.3) for 2-4 h, and change the solution 3 times. 3. 1.5% to 2.5% of the cellular fluid is used as the fixation medium.

3. 1.5%-2% potassium pyroantimonate-1% osmium acid fixed at 4℃ for 2 h. 4. pH 10 distilled water.

4. pH 10 distilled water rinsing 3 times × 10 min, in order to wash out the residual pyroantimonate.

5. Dewatering, embedding as usual, sectioning, staining or no staining. 6.

6. Electron microscopic observation.

Caveat

1. Potassium pyroantimonate dissolves slowly at room temperature, but rapidly when heated to 90-100°C. The results are not identical when potassium phosphate or potassium oxalate is applied.

2. The results are not identical when potassium phosphate or potassium oxalate is applied. Generally heart, liver, kidney, brain and other tissues, blood cells and cultured cells can be used.

3， pyroantimonate can also react with sodium, magnesium and other ions to form a precipitate, so we need to control the experimental conditions, so that pyroantimonate mainly reacts with calcium, less or no reaction with sodium and magnesium, with 1.5% to 2% of potassium pyroantimonate at 4 ℃ reaction is more appropriate (in this reaction conditions of the experiments for EDX spectroscopy, the results show that the precipitate does not contain sodium, magnesium, or very little).

4. Potassium pyroantimonate penetrates into the cells very slowly, if the tissue is fixed by perfusion, and then cut into thin slices with an oscillating slicer, the results are better.

5. Ionized and bound calcium are present in the cells, and the two can be interconverted. Theoretically, it is ionized calcium that reacts with phosphate (oxalate)-pyroantimonate, but this may not be the case in practice, and some of the bound calcium that is not very strongly bound may also react with phosphate (oxalate)-pyroantimonate to form a precipitate.

6. Staining after sectioning, for routine observation, may be done as for general staining. For spectroscopic analysis, it is not stained and, if necessary, may be fixed in 1% osmium acid and then in 2% potassium ferrous fluoride after osmium fixation to improve the contrast.

Common Problems

Source : Experimental Techniques and Applications of Immunohistochemistry

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