Experiments on live staining of mitochondria in cells

Summary

Cells carry out many kinds of life activities in the whole life process, which are realized by various inherent structures and their components in the cells, and it is of great significance to understand the life process of cells by truly reflecting the characteristics of these structures and components in different cells. Live staining is a method that can reflect the characteristics of the cell in its active state, and it is a staining method that utilizes certain non-toxic or less toxic stains to make certain structures or components within the cell appear in their natural state.

Principle

The basic principle of the mitochondrial vivo staining experiment is that the stain used for vivo staining should be specific and not affect or less affect the normal life activities of the cell. Live staining is usually used to show a specific structure in the cell, such as mitochondria, nucleus, etc., but also to determine the survival of the cell, the commonly used dyes are Janus green, neutral red and so on. Janus green B (Janus green B) is a specialized live stain for mitochondria. It is alkaline and fat-soluble, and can enter the cell through the cell membrane and bind to the negatively charged inner mitochondrial membrane through the positively charged chromophore groups in its structure. Mitochondria is an important place for energy metabolism in the cell, containing a variety of enzymes related to energy metabolism, in which the cytochrome oxidase on the inner membrane can keep the bound Janus green oxidized and blue, while in the surrounding cytoplasm the dye is reduced to colorless.

Operation method

Experiments on live staining of mitochondria in cells

Principle

The basic principle of the mitochondrial vivo staining experiment is that the stain used for vivo staining should be specific and not affect or less affect the normal life activities of the cell. Live staining is usually used to show a specific structure in the cell, such as mitochondria, nucleus, etc., but also to determine the survival of the cell, the commonly used dyes are Janus green, neutral red, etc.. Janus green B (Janus green B) is a mitochondrial stain, alkaline, fat-soluble, can cross the cell membrane and enter the cell, and through the structure of the positively charged chromophore binding to the negatively charged mitochondrial inner membrane. Mitochondria is an important place for energy metabolism in the cell, containing a variety of enzymes related to energy metabolism, in which the cytochrome oxidase on the inner membrane can keep the bound Janus green oxidized and blue, while in the surrounding cytoplasm the dye is reduced to colorless.

Materials and Instruments

Equipment:
Dissecting equipment, dissecting trays, flat dishes, slides, cover slips, pipettes, blotting filters, syringes, general optical microscope, rabbits.
Reagents:
① 0.9% Ringer's liquid (for mammals) (sodium chloride, 0.9 g; potassium chloride, 0.042 g; calcium chloride, 0.025 g; distilled water, 100 mL).
② 1/300 Janus Green B (Janus Green B 1.0 g, 100 mL of Ringer liquid, stored in a brown bottle, preferably before use); ① 1/300 Janus Green B (Janus Green B 1.0 g, 100 mL of Ringer liquid, preferably before use).

Move

The basic procedure of mitochondrial in vivo staining in cells can be divided into the following steps:
A. Take 1 rabbit, execute it by air embolization, place it in a dissecting tray, open the abdominal cavity quickly, and take a piece of thin liver tissue (2-3 ^{mm3 )} from the edge of the liver of the rabbit.
B. Place the piece of tissue on a petri dish and wash it with 0.9% Ringer's solution for 3 times to remove the blood.
C. Remove the Ringer's solution by sucking it with a piece of filter paper.
D. Add 1/300 Janus Green B staining solution and stain for 30 min.
E. Transfer the tissue block to a slide, pull it to pieces with forceps, remove the block and leave the cells behind.
F. Place 1 drop of Ringer's solution, cover the slide, and aspirate the excess liquid from the side of the slide with a piece of filter paper.
G. Observe under the microscope.

Caveat

1. Because this experiment is live staining, care should be taken to keep the specimen in a live state during the whole process of the experiment. When the cells die or begin to die, with the inactivation of enzymes, the cytoplasm and nucleus are also stained. When taking the material, it should be accurate and fast; when staining, the surface of the tissue block should be exposed to the staining solution, so that the enzymes of the intracellular mitochondria can fully carry out oxidation, maintain the oxidized state of the dye, and exert the staining efficacy.

2. Janus green has weak toxicity, and too long staining time may lead to the formation of vacuoles in mitochondria, so care should be taken in the operation.

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