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Tissue clearing kits and methods

A kit and reagent technology, applied in the biological field, can solve the problems of shaping and inability to realize biological tissue, etc.

Active Publication Date: 2022-03-22
GUANGDONG ZHANJIANG PROVINCIAL LAB OF SOUTHERN MARINE SCI & ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The main purpose of the present invention is to propose a tissue clearing kit and method, aiming to solve the technical problem that the prior art cannot realize the shaping of biological tissue

Method used

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  • Tissue clearing kits and methods
  • Tissue clearing kits and methods
  • Tissue clearing kits and methods

Examples

Experimental program
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Embodiment 1

[0043] In this embodiment, the small intestine tissues of C57 mice (fixed by 4% paraformaldehyde and 2 cm in length) were used, and three experimental groups (numbered as I, II, III) and three control groups (numbered as IV, V) were set up. , VI). Please refer to Table 1 for the composition and concentration of reagent 1 and reagent 2 in the experimental group, and the composition and concentration of the replacement reagent in the control group. In the three control groups, the glucose was replaced by mannose, fructose and sorbitol, respectively. In this embodiment, the tissue clearing method of the present invention is adopted. For the incubation temperature and time of the tissue, please refer to Table 2. See Table 3 for the experimental results.

[0044] Table 1 Reagent composition and concentration of experimental group and control group

[0045]

[0046] Table 2 The incubation temperature and time of the experimental group and the control group

[0047]

[0048...

Embodiment 2

[0053] The cleared tissue can be sequentially imaged and observed under confocal, two-photon and light-sheet microscopes. figure 1 Observation process of mouse small intestine tissue imaging, including dissection (Dissection), fixation (Fixation), staining (Staining), clearing (Clearing), LSCM imaging (Imaging), TPFM imaging (Imaging) and data analysis (Date Analysis) . The specific process is: after the mouse is anesthetized with pentobarbital sodium, 0.01M sodium citrate-phosphate buffer solution is perfused through the apex of the heart, until the blood in the mouse body is completely replaced, the required After the tissue was fixed overnight with 4% paraformaldehyde, it was sequentially soaked in the reagent 1 and the reagent 2 for clearing treatment, and finally imaged under a microscope.

[0054] In this embodiment, the reagent one includes: 30% m-xylylenediamine, 5% urea, and 10% glucose; the second reagent includes: 40% m-xylylenediamine, and 40% glucose. In the rea...

Embodiment 3

[0057] This embodiment compares the clearing effect of the method of the present invention and the existing clearing method on the tissue. The sample adopts the small intestine tissue of C57 mice, including the experimental group (the method of the present invention, PHCS) and three control groups (CUBIC, MACS, FOCM ). Three samples were selected for each group, and their light transmittances in the wavelength range of 400nm to 800nm ​​were measured respectively, and the average value was calculated.

[0058] image 3 Procedure and timing for processing small intestinal tissue for four clearing methods. Specifically, the experimental group (PHCS): soak the tissue in the reagent one (PHCS1, containing 30% m-xylylenediamine, 5% urea, and 10% glucose), incubate at 25°C for 12h to 24h, and then The tissue was soaked in the reagent 2 (PHCS2, containing 40% m-xylylenediamine and 40% glucose), and incubated at 4°C for 12h-24h. Control group (CUBIC): soak the tissue in Reagent-1 (c...

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Abstract

The invention discloses a tissue clearing kit and method, wherein the tissue clearing kit includes aromatic amine and glucose, and the aromatic amine and glucose are used to prepare reagent one and reagent two for tissue clearing treatment respectively; in reagent one , the mass ratio of aromatic amine is 25-40%, and the mass ratio of glucose is 5-10%. In the second reagent, the mass ratio of aromatic amine is 30-45%, and the mass ratio of glucose is 30-45%. The tissue clearing kit of the present invention can realize the clearing treatment of tissues (especially tubular and cavity-shaped tissues), and shape the cleared tissues, so that the tissues can be used for preparing frozen sections or tissue specimen slices.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a tissue clearing kit and method. Background technique [0002] With the rapid development of life science technology, the use of biological tissue transparency technology to obtain three-dimensional image information of biological tissue has become a research hotspot. At present, transparency technologies such as FOCM, SCALE, CUBIC, SEEDB, and MACS have been widely used in the field of life sciences. However, the tissues (especially tubular and cavity-shaped tissues) treated with the existing clearing reagents are relatively loose and soft, and difficult to shape, so that the tissues cannot be modified or sliced. [0003] The above content is only used to assist in understanding the technical solution of the invention, and does not mean that the above content is admitted as prior art. Contents of the invention [0004] The main purpose of the present invention is to propose a ti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/36G01N21/64
CPCG01N1/36G01N21/6456G01N21/6458
Inventor 吴科锋李茜叶华罗辉朱宇珍戚怡
Owner GUANGDONG ZHANJIANG PROVINCIAL LAB OF SOUTHERN MARINE SCI & ENG
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