Fluorescent labeling and observation of plant organelles

Summary

Plant cells contain abundant organelles, including vesicles, plastids, mitochondria, Golgi apparatus, ribosomes, endoplasmic reticulum, microtubules and microfilaments, and nuclei, etc. Among them, vesicles and plastids are organelles specific to plant cells. With the deepening of cell biology research, most of the organelles in plant cells can be labeled by specific fluorescent dyes, which greatly facilitates the exploration of intracellular life activities.

Currently, many organelles in plant cells can be specifically labeled by dyes. Some dyes can pass through the cell membrane and are used to label specific organelles in living cells, while others cannot pass through the cell membrane and can only label specific organelles in dead cells, and some commonly used organelle dyes are listed in the following table (Table 26-1).

Table 26-1 Fluorescent Dyes and Staining Principles of Commonly Used Organelles

Organelle

dyestuff

Live/Dead Cells

Staining Principle

nucleus (of a cell)

DAPI (4',6-diamidino-2-phenylindole)

Live/dead cells

DAPI is a fluorescent dye that can penetrate the cell membrane and bind to the double-stranded DNA in the nucleus of the cell to play the role of labeling, generating fluorescence that is more than 20 times stronger than DAPI itself, and you can see the blue fluorescent cells under the microscope.

Vesicles

Neutral red

live cell

Neutral Red is a weakly alkaline pH indicator that changes color from pH 6.4 to 8.0 (from red to yellow). In a neutral or slightly alkaline environment, the living cells of plants can take up a large amount of Neutral Red and transport it to the vesicles. Since the vesicles are generally acidic, the Neutral Red that enters the vesicles dissociates a large number of cations and takes on a cherry red color. In this case, the protoplasm and cell walls are generally not colored; in dead cells due to the degeneration of protoplasm coagulation, the cytosol can not be maintained in the vesicle, therefore, after staining with neutral red, does not produce vesicle coloring phenomenon, on the contrary, the cations of the neutral red, but with a certain degree of negatively charged protoplasm and nucleus binding, and make the protoplasm and nucleus stained.

Mitochondria

Janus green B (Janus green B)

Live cells

Janus green B staining is due to the action of a cytochrome oxidase system in the mitochondria, which keeps the dye oxidized to a bluish-green color at all times; in the surrounding cytoplasm the dye is reduced to a colorless chromophore

Microfilaments

Ghost pen cyclic peptide (phalloidin)

Live/Dead Cells

Ghostpen cyclic peptide binds specifically to microfilaments, stabilizing microfibrils and inhibiting their function. Fluorescently labeled Ghost Pen Cyclic Peptide specifically displays intracellular microfilaments.

Operation method

Fluorescent labeling and observation of plant organelles

Materials and Instruments

Root tips of soybean seedlings (mung beans or rice are also acceptable) are cultivated on moist filter paper in petri dishes and allowed to germinate until the radicle elongates more than 1 cm. Epidermal cells of onion bulbs.
Apparatus:
① microscope
① Microscope ② Tweezers
① microscope ② tweezers ③ slide
① microscope ② tweezers ③ slide ④ coverslip
Reagents:
① Ringer's solution
① Ringer's solution ② 1% Neutral Red solution
① Ringer's solution ② 1% Neutral Red solution ③ 1/3000 Neutral Red staining solution
① Ringer's solution ② 1% Neutral Red solution ③ 1/3000 Neutral Red staining solution ④ 1%, 1/5000 Janus green B staining solution
⑤ 4% paraformaldehyde solution (freshly prepared)
⑥ DAPI staining solution

Move

The basic process of fluorescent labeling and observation of plant organelles can be divided into the following steps:

(i) Neutral red staining and observation of plant cell vesicles

1. Observe the vesicles in the root tip cells of soybean seedlings.

Use a double-sided blade to cut a longitudinal section of the root tip of a newborn soybean seedling (1~2 cm long), put it into 1/3000 neutral red staining solution in the center of the slide, and stain it for 5~100 min. Aspirate off the neutral red staining solution, replace it with Ringer's solution, and then cover the slide for observation. There were many round vesicles of different sizes stained with rose-red color in each growth point cell. If you observe the differentiated cells, you can see larger vesicles, fewer in number, sometimes only one huge light red vesicle. 2.

2. Observe the vesicles of the epidermal cells of onion bulb.

Tear the epidermal cells of onion bulb and put them into 1/3000 neutral red staining solution in the center of the slide and stain for 5~10 min. Aspirate off the neutral red staining solution, replace it with Ringer's solution and cover the slide for observation.

(ii) Staining and observation of mitochondrial Jamis green B in plant cells.

Tear off a small piece of epidermis of onion bulb with a spatula, put it in 1/5000 Janus green B staining solution, it usually takes 30 min. move the stained material to the center of the slide (the whole stained root tip torn off a piece of epidermis), put a little drop of Ringer's solution, cover with a coverslip, and then observe. Under the microscope, the center of the epidermal cells of the onion bulb is occupied by a huge vesicle, the nucleus is squeezed to the side, and the mitochondria in the cytoplasm are stained blue-green and appear as granules or lines.

(iii) Staining and observation of nucleus of plant cells

Tear off a small piece of epidermis of onion bulb with a shackle and fix it in 4% paraformaldehyde solution for 1 h. Move the fixed material to a small dish and wash it with PBS 3 times, each time for 5 min, then move the material to the center of slide, drop a little bit of DAPI staining solution, cover the coverslip and observe. Under the microscope, the nuclei of the epidermal cells of onion bulbs were stained blue and spherical.

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