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Staining method for living cell imaging

A technology of living cells and staining methods, which is applied in the field of cell staining and double staining using fluorescent biomarkers, which can solve the problems of inability to observe the shape of the nucleus, the inability to observe the cell shape, and the long staining process.

Active Publication Date: 2021-09-07
张慧敏
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when living cells are observed by fluorescence microscopy, these staining agents can only be absorbed by the cell body or by the nucleus when they are absorbed by a single cell.
When living cells are stained with fluorescence and observed under a fluorescence microscope, acridine yellow staining imaging can observe cell nucleus morphology, but cell morphology cannot be observed, and sodium fluorescein staining imaging can observe cell morphology, but cell nucleus morphology cannot be observed
When performing microscopic observation, only the shape of the nucleus or the shape of the cell can be observed, which does not meet the needs of observing the nucleus and the shape of the cell at the same time when observing the cell
[0012] To sum up, the current fluorescent staining method commonly used in live cell imaging analysis needs to fix the cells in practical application, and the use process is cumbersome. The staining process takes a long time, is non-specific, and has insufficient contrast. Disadvantages such as cell nucleus morphology

Method used

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  • Staining method for living cell imaging

Examples

Experimental program
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Effect test

Embodiment 1

[0081] Example 1: Tissue staining by sodium fluorescein and methylene blue double staining

[0082] 1.1 Mouse kidney staining

[0083] Using mice for tissue staining, the staining process is as follows:

[0084] 1. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and the anesthesia dose was 8–9 mL / g.

[0085] 2. Remove the body hair on the back of the mouse, cut the epidermis to expose the kidney, remove the kidney completely, fix the surface of the kidney with a blade, and remove the capsule on the surface of the kidney with scissors and tweezers under a microscope.

[0086] 3. Use a cotton swab to stop bleeding, apply 0.25% sodium fluorescein to the surface of the kidney for 2 minutes, wash it with normal saline three times; then apply 1% melanin staining solution for 2 minutes, wash it with normal saline three times after the time is up, Then place it under a microscope for observation at a wavelength of 470nm.

[0087] The results in Figur...

Embodiment 2

[0095] Example 2: Effects of double staining with different concentrations of fluorescein sodium and methylene blue staining

[0096] Use porcine kidney for tissue staining, the staining process is as follows:

[0097] 1. Purchase some fresh pig kidneys and refrigerate them at 4°C for later use.

[0098] 2. Clean the fresh pig kidney with clean water, and remove the membrane on the surface of the kidney with scissors and tweezers under a microscope.

[0099] 3. Apply and stain the surface of the kidney with 0.1%, 0.25%, 0.5%, and 1% fluorescein sodium for one minute, then wash with normal saline three times; then stain with 0.5%, 1%, 2%, and 3% methylene blue respectively Apply and dye the surface of the liquid for one minute. After the time is up, wash it with physiological saline three times, and then observe it under a microscope with a wavelength of 470nm.

[0100] Figure 3 is a comparison of the imaging effects of pig kidneys stained with different concentrations of sod...

Embodiment 3

[0102] Example 3: 5-ALA and Acridine Yellow Double Staining Method for Tissue Staining

[0103] Pig liver was used for tissue staining, and the staining process was as follows:

[0104] 1. Purchase some fresh pig livers and refrigerate them at 4°C for later use.

[0105] 2. Clean the fresh pig liver with clean water, and remove the surface film of the liver with scissors and tweezers under a microscope.

[0106] 3. Dye the surface of the liver with 0.05% 5-ALA for 3 minutes, wash it with normal saline three times; then apply and dye the surface with 1% acridine yellow staining solution for 2 minutes, wash it with normal saline three times after the time is up, and then put it in Observed under a microscope at a wavelength of 635nm.

[0107] Figure 4 The imaging results of double-staining with 5-ALA and acridine yellow for pig liver can clearly see the outline of the stained liver cells and the shape of the nucleus.

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Abstract

The invention provides a fluorescent staining method for living cell imaging, cells are subjected to double staining through the first fluorescent biomarker and the second fluorescent biomarker, clear fluorescent cell images are detected and displayed through a fluorescence microscope, and according to the obtained images, the cell morphology can be observed while the cell nucleus morphology can be observed.

Description

technical field [0001] The invention relates to a cell staining method, in particular to a double staining method using fluorescent biomarkers, and belongs to the field of biotechnology. Background technique [0002] The combination of fluorescence staining method and fluorescence microscopy technology has made fluorescence imaging widely used in the detection of biologically active substances and cell imaging, among which cell imaging technology is an important research method in the field of life science technology. Compared with other techniques, fluorescent staining has the advantages of high sensitivity, high selectivity, simple operation, and sensitive response. It is a highly sensitive visual analysis technique widely used in live cell analysis. [0003] Fluorescence is a "photoluminescence" luminescence phenomenon. When a substance at room temperature is irradiated by incident light of a specific wavelength, it absorbs light energy and enters an excited state, and i...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N1/30
CPCG01N21/6428G01N21/6486G01N1/30G01N2021/6439
Inventor 张慧敏
Owner 张慧敏
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