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Embedding agent, embedding method and application of light-transparent biological tissue

A biological tissue and embedding agent technology, applied in the field of biological imaging, can solve the problems of incompatibility, inconvenient storage of samples, and difficulty in maintaining fluorescence signals for a long time, and achieve the effects of rapid imaging, long fluorescence signal time, and easy availability of reagents.

Inactive Publication Date: 2018-12-07
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] Aiming at the above defects or improvement needs of the prior art, the present invention provides an embedding agent, method and application of light-transparent biological tissue, the purpose of which is to select an embedding agent with a suitable refractive index for Embed biological tissue or biological tissue labeled with exogenous fluorescent dye to realize the transparency of biological tissue, while both endogenous fluorescence and exogenous fluorescence are kept good, thus solving the problem of the existing chemical reagents for transparent biological tissue of liquid reagents and In the clearing method, there are technical problems that fluorescent protein labeling and fluorescent dye labeling are incompatible, sample storage is inconvenient, and fluorescent signals are difficult to maintain for a long time

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Embodiment 1 and 2 and 4

[0061] An embedding method of an embedding agent for biological tissue, which is a medium and low temperature solidification embedding method, figure 1 It is the process of resin embedding biological tissue transparent method, such as figure 1 shown, including the following steps:

[0062] (1) Biological tissue pretreatment: firstly, after perfusion, the mouse brain was taken out and fixed in 4% paraformaldehyde (PFA) for 24 hours; then rinsed with PBS solution for 3 times, 8 hours each time. Then use tetrahydrofuran / distilled aqueous solution to dehydrate according to the gradient at 4°C, 50% tetrahydrofuran / 2 hours+75% tetrahydrofuran / 2 hours+95% tetrahydrofuran / 2 hours+100% tetrahydrofuran / 2 hours+100% tetrahydrofuran / 4 hours;

[0063] (2) Permeation: then immerse the dehydrated rat brain in the prepared resin monomer at 4°C in a dark environment for 2 hours, then change the liquid, and then immerse for 2 days;

[0064] (3) Curing: The embedding agent is as shown in Table...

Embodiment 2

[0067] An embedding method of an embedding agent for biological tissue, comprising the steps of:

[0068] Step (1) and step (2) are the same as embodiment 1.

[0069] (3) curing: the embedding agent is as shown in table 1, by weight, the mixture of 95 parts of acrylate monomer phenyl methacrylate shown in table 1 and 5 parts of crosslinking agent tetraethylene glycol diacrylate After mixing with 1 part of initiator azobisisoheptanonitrile according to the weight ratio of 99:1, blow with nitrogen gas at a certain flow rate for 30 minutes, and store the mixed resin monomer in a refrigerator at -30°C for later use;

[0070] Finally, put the infiltrated mouse brain in the capsule and add the mixed spare resin monomer, then put it in a vacuum oven, and cure it according to the set curing conditions, and wait for it to slowly drop to room temperature and cool down, then the imaging process can be carried out , wherein the curing conditions and specific steps are: curing at an initi...

Embodiment 3

[0072] (1) Biological tissue pretreatment: First, take out the rat brain after perfusion and place it in 4% paraformaldehyde (PFA) and fix it for 24 hours; then slice the rat brain with a vibrator, and then rinse it with PBS solution for 3 times, 8 hours each time. The fixed and rinsed biological tissues were immunohistochemically labeled with Alexa 488, an organic fluorescent dye molecule. First, use 20% DMSO and 0.2% Triton X-100 PBS solution to perforate 100 micron mouse brain tissue slices for 12 hours, then add 10% serum to block for 12 hours, rinse with PBS for 1 hour / 3 times, and then follow Add the primary antibody at a ratio of 500:1 and incubate with shaking at 37°C for 2 days in the dark, then incubate with shaking at 37°C for 2 days in the dark and then add the secondary antibody at a ratio of 800:1 and incubate at 4°C for 8 hours in the dark and then use PBS Rinse for 1 hour / 3 times.

[0073] (2) Permeation: After fluorescent dye immunohistochemistry, the brain ...

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Abstract

The invention discloses an embedding agent for light-transparent biological tissue and an embedding method thereof. The embedding agent is a high refractive index acrylate resin, including two components A and B; component A includes 90 to 100 parts by weight of acrylate resin monomer, 0 to 10 parts of diacrylate crosslinking agent; B component includes by weight, less than or equal to 10 parts of alkenyl polymerization initiator; the weight ratio of A component and B component is between 90:10 and 999:1. By adopting the embedding agent of the present invention to embed biological tissue, the degree of light transparency of biological tissue is high, the method is simple and easy to operate, the fluorescence retention effect of endogenous fluorescent protein and exogenous fluorescent dye is good, and the embedding success rate is high, which can Applied to light-sheet fluorescence microscopy and wide-field fluorescence microscopy, and potentially electron microscopy.

Description

technical field [0001] The invention belongs to the field of biological imaging, and more specifically relates to an embedding agent, embedding method and application of light-transparent biological tissue. Background technique [0002] Studying the network structure of brain nerves is of great significance for the treatment of human mental diseases and the study of human cognition and emotion. Obtaining the 3D neural network structure of the whole brain and reaching the resolution of synaptic level is a cross-field for fluorescence imaging. It is a huge challenge, and it can also promote technological progress in the entire field of science and technology in the future, especially the study of the relationship between brain structure and function from the perspective of complete system organization and function. It is still very difficult to study the structural information of a single molecule of a biological tissue in a complete biological system. For optical imaging of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F220/30C08F220/18C08F222/14G01N1/30
CPCG01N1/30C08F220/18C08F220/30C08F220/301C08F222/104C08F222/102
Inventor 曾绍群周宏福刚亚栋熊雨苗刘秀丽
Owner HUAZHONG UNIV OF SCI & TECH
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