Experiments on live staining of mitochondria

Summary

Mitochondria are an important organelle within the cell and are the site of cellular respiration. The energy required for various cellular activities is mainly supplied by mitochondrial respiration.

Operation method

basic program

Principle

Live staining is a method of applying non-toxic or less toxic stains to realistically visualize certain structures in living cells with little effect on cellular activity. Janus Green B is a specific live stain for mitochondria. Cytochrome oxidase in the mitochondria keeps the dye oxidized to a blue-green state, while in the surrounding cytoplasm the dye is reduced to a colorless state.

Materials and Instruments

Rabbits
Microscopes Surgical Equipment Dissecting Plates Flatware Slides Coverslips Pipettes Syringes Straws
Janus Green B Stain Ringer's Solution

Move

I. Methodology
1. Execute the rabbit by air embolization, place it in a dissecting tray and quickly open the abdominal cavity.

2. Take a small piece (2~3 mm³ ) of the thinner liver tissue at the edge of the rabbit's liver and put it into a flat dish containing Ringer's solution to wash away the blood (press gently with forceps).

3. Remove the Ringer's solution with a pipette and add 1/300 of Janus Green B dye to the dish, so that the upper surface of the tissue block is exposed to the outside of the dye, so that the enzyme system of the mitochondria in the cell can be fully oxidized, which is conducive to maintaining the oxidized state of the dye and coloring the mitochondria.

4. When the edge of the tissue block is stained to a basket color, it is ready, usually need to be stained for 30 minutes.
5. After staining, move the tissue block onto a carrier plate and pull the block apart with tweezers; some cells or groups of cells will detach from the block.
6. Remove slightly larger pieces of tissue so that free cells or cell populations remain on the slide, add a drop of Ringer's solution, cover the slide, and absorb excess water.
II. Results
Microscopic observation showed that many mitochondria in the liver cytoplasm were stained blue-green and granular.

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