Radiographic autoradiography experiment

Summary

The radioisotope itself can record the site and intensity of its presence in close contact with photographic latex, showing accurately the localized relationship between form and function. Culturing. Radiographic autoradiography is a method of showing the distribution, quantification, and localization of radioactivity in a specimen or sample by using the sensitizing effect of ionizing radiation from a radioisotope on a nucleus latex.

Principle

The principle of radiographic self-development is that the radioactive isotope of the material under study releases rays, which act on the photographic emulsion to make the silver halide in the photographic emulsion light-sensitive to form a latent image, and then by the action of the developer, the light-sensitive silver halide is restored to the black silver particles, and the silver salts that are not light-sensitive are removed by the fixation, leaving a clear and unambiguous image in the emulsion.

Operation method

Radiographic autoradiography experiment

Principle

Cultured cell radiography is based on the principle that the radioactive isotope of cultured cells releases rays, which act on the photographic emulsion to sensitize the silver halide in the photographic emulsion to form latent images, and then, through the action of the developer, the sensitized silver halide is restored to black silver particles, and the un-sensitized silver salts are removed by fixation, leaving a clear and distinct image in the emulsion.

Materials and Instruments

Equipment:
① Cultured cells
② Equipment for cell passaging culture
③ 3.5 cm diameter plastic petri dish (or 50 ml culture flask)
④ 2.2 cm x 2.2 cm cover sheet (or cut to 6 mm x 40 mm and place in culture flask)
⑤ Exposure lead box or black plastic box
⑥ Electromagnetic stirrer
⑦ Thermostat (adjustable temperature for drying latex)
⑧ Carrier sheet
⑨ Neutral gum
⑩ Darkroom and appropriate equipment
Reagents:
① Isotope markers (e.g. 3 H-TdR, 3 H-UdR, etc.)
② liquid emulsion (commonly used nuclear IN number)
③ Developing solution
Developer ④ Fixer
⑤ Methanol
⑤ Methanol

Move

The basic procedure of cultured cell radioautography can be divided into the following steps:
(1) Binding of isotope marker to cells
A. Dilute the isotope marker with culture medium, and the common concentration of isotope marker for cultured cell radioautography is 0.1-2 μ Ci/ml.
B. Discard the medium from the cultured cells, and wash with Hanks buffer 1 time. The medium containing the isotope was washed once with Hanks buffer, and then added to the above medium containing the isotope. Generally, add 3 ml of culture medium to a 50 ml culture flask.
C. Place the above cells in a constant temperature oven at 37 ℃ for a certain period of time (the incubation time depends on the requirements of the experiment and the isotope type and concentration, etc., but generally speaking, incubation for 1 to 4 h is sufficient if the isotope is 3 H-TdR at a concentration of 1 μ Ci/ml), and then discard the culture medium and swish the cells 3 times in Hanks buffer, so as to remove the radioactive substances that have not been mixed into the cells.
(2) Fixation
is carried out according to conventional cytological methods, but the use of fixative containing heavy metal ions should be avoided to avoid increasing the number of background particles. It is recommended to use pure methanol for fixation for more than 30 min.
(3) Coating latex
A. Preparation of latex Under the red light in the dark room, take out the nuclear IN latex from the opaque container and put it in a 10 ml small beaker, put the small beaker on the electromagnetic stirrer, put in 1 small glass rod with clean iron core, turn on the electromagnetic stirrer to make the latex gelatinized, and for the purpose of dilution, add appropriate amount of additional liquid or triple-distilled water, and stir it for a few minutes to make the latex and the additional liquid mix well. If there is no electromagnetic stirrer, preheat triturated water at 40 ℃, then add equal or 2/3 amount of nuclear IN latex and stir well with a clean glass rod, avoiding air bubbles.
B. Remove the small cover slip (or the square cover slip in the Petri dish) with cultured cells, stick one end of the cover slip with adhesive tape on the carrier sheet (note: make the side with cells face up, this side is rougher in appearance), and then put 1 drop of latex on the cover slip with cells. Then put 1 drop of latex on the cover slip with cells on it, and spread the latex gently with a pipette.
C. Place the above latex-coated slide upright on a sectioning frame, so that the latex becomes a thin layer as much as possible, and then blow it dry with a small fan at a low speed.
D. When the latex is dry and hard (10-20 min), put the slides into an exposure box with a desiccant (silica gel or calcium chloride is commonly used), and then wrap the box tightly with a black paper, marking it with the marking of isotope specimen, and then put it into a refrigerator at 4 ℃ or at room temperature for exposure. Expose the specimen at room temperature.
(4) Exposure
The exposure time depends on the radioactivity of the specimen, the isotope dose and the experimental requirements. In order to elucidate the precise localization of the isotope in cells and tissues, the exposure time should be short; in order to show the localization of a small amount of radioisotope, the exposure time should be long. For isotopes with long half-lives, the exposure time should be increased accordingly for specimens with small activity. In order to select the optimal exposure time, reference can be made to similar work, and experimental exposures should generally be carried out to determine the optimal exposure time.3 H, C, 35 S markers are usually exposed for 3-7 d. The exposure time should be longer than that for the same specimen.
(5) Developing and fixing
The developing and fixing time varies with the exposure time and isotope dose. In the general photographic developing solution, develop for 3~5 min at 18~20 ℃, then place in 1% acetic acid solution for 1~3 min, and then transfer to the fixing solution for about 30 min.
(6) Staining
Rinse the specimen with tap water continuously for several minutes (do not run the water too fast or too large), then wash it once with triple-distilled water, dry it and then stain it with Giemsa staining (HE staining can also be used).
(7) Sealing
After staining and drying, seal the specimen with neutral gum, and observe and photograph under an oil microscope.

Caveat

1. There are various methods of applying latex, the principle being that it should be applied uniformly and, in addition, as thinly as possible.

2. Starting from the coating of emulsion, the process shall be carried out in a dark room until the finalization is completed before seeing light.

3. Disposal of isotope waste must be carried out in strict accordance with the requirements and must not pollute the environment.

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