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Staining solution for fluorescent staining of covalent bonds of expanded biological tissues and application of staining solution

A technology for fluorescent dyeing and biological tissue, applied in the field of dyeing liquid, can solve the problem of high cost of dyes and achieve the effect of cheap raw materials

Pending Publication Date: 2022-02-08
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantage of the existing NHS ester dyeing method with fluorescent molecules is the high cost of dyes required for dyeing

Method used

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  • Staining solution for fluorescent staining of covalent bonds of expanded biological tissues and application of staining solution
  • Staining solution for fluorescent staining of covalent bonds of expanded biological tissues and application of staining solution
  • Staining solution for fluorescent staining of covalent bonds of expanded biological tissues and application of staining solution

Examples

Experimental program
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Effect test

Embodiment 1

[0023] The invention performs staining and three-dimensional imaging on the expanded mouse lung tissue.

[0024] (1) Tissue sampling: a mouse lung tissue block with a size of 3mm×3mm×3mm was fixed in 4% paraformaldehyde and 20% acrylamide for 1 day;

[0025] (2) Swelling and transparent treatment of tissue samples: the fixed tissue is placed in a hydrogel monomer solution (the mass concentration of each component is as follows: 20% acrylamide, 0.05% methylenebisacrylamide, 4% paraformaldehyde, 10 % sodium acrylate, 0.1% VA-044 polymer initiator, and the solvent is phosphate buffer) at 4°C for 1 day, then put the hydrogel and tissue at 50°C to polymerize into a gel for 4 hours, and the gelled tissue Place in protein denaturing solution (50mM Tris, 200mM Sodium Dodecyl Sulfate, 200mM Sodium Chloride, pH 9.0) at 70°C for 24 hours, then place at 90°C for 24 hours, then put the sample into PBS buffer and 0.1% Triton X-100 for 4 hours;

[0026] (3) Covalent bond fluorescence stain...

Embodiment 2

[0030] The invention performs staining and three-dimensional imaging on the expanded mouse kidney tissue.

[0031] (1) Tissue collection: a mouse kidney tissue block with a size of 3mm×3mm×3mm was fixed in 4% paraformaldehyde and 20% acrylamide for 1 day;

[0032](2) Swelling and transparent treatment of tissue samples: the fixed tissue is placed in a hydrogel monomer solution (the mass concentration of each component is as follows: 20% acrylamide, 0.05% methylenebisacrylamide, 4% paraformaldehyde, 10 % sodium acrylate, 0.1% VA-044 polymer initiator, and the solvent is phosphate buffer) at 4°C for 1 day, then put the hydrogel and tissue at 50°C to polymerize into a gel for 4 hours, and the gelled tissue Place in protein denaturing solution (50mM trishydroxymethylaminomethane, 200mM sodium dodecyl sulfate, 200mM sodium chloride) at 70°C for 24 hours, then place at 90°C for 24 hours, put the sample into PBS buffer and Wash in 0.1% Triton X-100 for 4 hours;

[0033] (3) Covalen...

Embodiment 3

[0037] Swelling clearing, staining, and three-dimensional imaging of ischemia-reperfusion mouse kidney tissue.

[0038] (1) Tissue samples: samples were collected from mice 6 hours after ischemia-reperfusion. Mouse kidneys were cut into 3mm×3mm×3mm tissue pieces and fixed in 4% paraformaldehyde and 20% acrylamide for 1 day. The control group was normal mice;

[0039] (2) Swelling and transparent treatment of tissue samples: the fixed tissue is placed in a hydrogel monomer solution (the mass concentration of each component is as follows: 20% acrylamide, 0.05% methylenebisacrylamide, 4% paraformaldehyde, 10 % sodium acrylate, 0.1% VA-044 polymer initiator, and the solvent is phosphate buffer) at 4°C for 1 day, then put the hydrogel and tissue at 50°C to polymerize into a gel for 4 hours, and the gelled tissue Place in protein denaturing solution (50mM trishydroxymethylaminomethane, 200mM sodium dodecyl sulfate, 200mM sodium chloride) at 70°C for 24 hours, then place at 90°C fo...

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Abstract

The invention discloses a staining solution for fluorescent staining of a covalent bond of an expanded biological tissue and application of the staining solution. The staining solution is prepared by dissolving rhodamine B and EDC into a phosphate buffer solution with the pH value of 7.4, the concentration of the rhodamine B is 7mM, and the concentration of the EDC is 1M. The dyeing method comprises the following specific steps: soaking a fixed expanded biological tissue in a hydrogel monomer solution at 4 DEG C for 1 day, polymerizing the hydrogel and the tissue at 50 DEG C to form gel for 4 hours, soaking the gel-formed tissue in a protein denaturation solution at 70 DEG C for 24 hours, soaking the tissue at 90 DEG C for 24 hours, and cleaning; soaking the sample in the staining solution at room temperature, oscillating for 48 hours, then cleaning the sample in a protein denaturation solution at 70 DEG C for 24 hours, and then cleaning the sample; and finally, carrying out nucleus dyeing and three-dimensional imaging. The method has the advantage that the dyeing cost can be greatly reduced.

Description

technical field [0001] The invention relates to a dyeing solution, in particular to a dyeing solution for covalent bond fluorescent dyeing of expanded biological tissue and its application. Background technique [0002] Three-dimensional imaging of biological tissues is gradually becoming a new method for studying histopathology because it has higher information content than traditional two-dimensional imaging. Expansion microscopy is a super-resolution microscopy technique developed since 2015. This method physically expands the sample (expands about 4 times in each dimension) by anchoring the protein in the biological sample in an expandable hydrogel. ) to improve the imaging resolution. Expansion microscopy has the unique advantage of not being limited by complex optical instruments. At the same time, the purpose of tissue transparency is achieved through protein denaturation and phospholipid removal during the swelling process. [0003] In 2020, Edward Boyden experime...

Claims

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Application Information

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IPC IPC(8): G01N1/30G01N21/84
CPCG01N1/30G01N21/84G01N2001/302
Inventor 严智洋李航远李泉鲁晶晶徐惠中
Owner SUZHOU UNIV
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