WO2020019670A1 - Treating liquid composition, kit, and biological organ transparentizing and immune labeling method - Google Patents

Treating liquid composition, kit, and biological organ transparentizing and immune labeling method Download PDF

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WO2020019670A1
WO2020019670A1 PCT/CN2018/124585 CN2018124585W WO2020019670A1 WO 2020019670 A1 WO2020019670 A1 WO 2020019670A1 CN 2018124585 W CN2018124585 W CN 2018124585W WO 2020019670 A1 WO2020019670 A1 WO 2020019670A1
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antibody
organs
transparent
treatment liquid
molecular weight
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PCT/CN2018/124585
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French (fr)
Chinese (zh)
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钟凯
徐金勇
汪增荣
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中国科学院合肥物质科学研究院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

  • the invention relates to a method for transparentizing and immunolabeling a whole organ of a biological tissue, in particular to a treatment solution and a method for directly or indirectly immunofluorescently marking a whole organ such as brain, spinal cord, articular cartilage, and organs while transparentizing.
  • the method of tissue transparency can realize large-scale high-resolution detection and three-dimensional structure reconstruction of tissue morphology in the case of non-destructive tissue. Compared with traditional tissue sectioning and staining methods, it has a significant improvement. Of all the tissue transparency methods currently developed, only part of the tissue transparency scheme allows for overall organ immunolabeling, such as iDisco, Clarity, Cubic, ACT-presto, etc.
  • Clarity and Cubic both remove the cell membrane and allow macromolecules to enter, but the process is tedious and takes nearly 1-2 weeks; iDisco uses organic solvents, has poor fluorescence compatibility, tissues become hard and brittle, and the pungent odor is harmful to the human body Harmful; ACT-presto relies on pressure, requires special equipment, and requires a complicated pre-treatment process before immunolabeling.
  • the invention aims to provide a simple and efficient technical solution for the whole organ or tissue to be transparent and immunolabeled.
  • the present invention provides a treatment liquid composition including:
  • a fluorescently labeled antibody, and a volume ratio of the fluorescently labeled antibody to the clearing treatment solution is 1:10 to 1: 500,
  • the transparent treatment liquid includes:
  • Non-ionic surface and surfactant includes koji with a volume ratio of 5: 3 to 1: 5
  • the sugar is at least one of fructose or sucrose.
  • Triton includes Triton X-100 and Triton X-114.
  • Tween includes Tween 20, Tween 40 and Tween 60.
  • each 100 ml of the clearing treatment liquid contains: 20-30 g of urea, 10-15 ml of non-ionic surfactant, 40-60 g of sugars, 0-20 ml of ammonia, 0-2 ml of anti-bacterial Oxidant, the balance is water.
  • the clearing treatment liquid is a series of treatment liquids, wherein as the sugar content increases, the urea content also increases, or as the sugar content decreases, the urea content also decreases.
  • the antibody includes at least one of the following: anti-AFP antibody, with a molecular weight of about 65 kD, preferably in a concentration range of 5-25 ⁇ g / ml; anti-Survivin antibody, with a molecular weight of about 16 kD, preferably in a concentration range It is 5-25 ⁇ g / ml; and anti-GFP antibody, the molecular weight is about 27kD, and the concentration range is preferably 10-100ug / ml.
  • the present invention also provides a treatment solution kit, comprising:
  • the first part includes: a transparent treatment liquid and a primary antibody mixed with the transparent treatment liquid in a volume ratio of 1:10 to 1: 500; and
  • the second part includes: a transparent solution and a fluorescently labeled secondary antibody mixed with the transparent solution in a volume ratio of 1:10 to 1: 500,
  • the clearing treatment liquid is the aforementioned clearing treatment liquid.
  • the primary antibodies in the treatment solution kit include:
  • anti-mTOR primary antibody anti-mammalian rapamycin target protein
  • anti-mTOR primary antibody anti-mammalian rapamycin target protein
  • anti- ⁇ -III Tubulin primary antibody (anti- ⁇ -III tubulin antibody), molecular weight of about 55kD, preferably in a concentration range of 10-50 ⁇ g / ml;
  • anti-NeuN primary antibody anti-neuron-specific nuclear protein antibody
  • molecular weight 46-55kD preferably in a concentration range of 10-200 ⁇ g / ml
  • anti-IIcollagen primary antibody (anti-type II collagen antibody), molecular weight about 142kD, mouse origin, preferably used in the concentration range of 10-200 ⁇ g / ml,
  • the fluorescently labeled secondary antibody is a fluorescently labeled immunoglobulin (IgG) capable of specifically binding to the primary antibody (anti-mouse or anti-rabbit).
  • IgG fluorescently labeled immunoglobulin
  • the present invention also provides a method for transparentizing biological organs and performing immunolabeling at the same time, including soaking biological tissues and organs with the above-mentioned treatment liquid composition; preferably, soaking at 37 ° C for 1-7 day.
  • the biological tissue organs are immersed in the treatment solution composition, and after the tissue is cleared, the tissue is washed with an antibody-free clearing treatment solution to remove unbound antibodies.
  • the present invention also provides a method for transparentizing biological organs and simultaneously performing immunolabeling, which includes treating biological tissues and organs with the above-mentioned treatment solution kit, wherein
  • the second part is used to soak the biological tissues and organs, preferably at 37 ° C for 1-3 days.
  • biological tissues and organs are first immersed in a primary antibody solution diluted with a clearing treatment solution. After the tissue is cleared, the clearing treatment solution is used to remove unbound primary antibodies; Soak biological tissues and organs with the fluorescently labeled secondary antibody solution diluted in the treatment solution. After about 1-3 days, wash with a clearing treatment solution without antibodies to remove unbound secondary antibodies.
  • the biological tissue organs include brain, spinal cord, articular cartilage, and organs (including tumor-bearing organs).
  • the biological tissues and organs include tissues such as mouse brain, rat brain, mouse spinal cord, and articular cartilage. Depending on the size of the tissue mass, they are immersed in a constant temperature at 37 ° C for 1-7 days.
  • the present invention provides a new method for direct or indirect immunofluorescence of tissues at the overall organ scale. This method realizes the immunofluorescence labeling on the whole organ scale while the tissue is transparent.
  • the entire operation process is simple, and no special experimental device is required; the immunofluorescence effect of the method of the present invention is determined, and it can be performed at a relatively fast speed.
  • FIG. 1 is a two-photon microscope image of a mouse brain specimen in Experiment 1 after direct immunofluorescence of whole brain of the treatment solution composition of the present invention.
  • FIG. 2 is a two-photon microscope image of a mouse tumor-bearing liver specimen in Test 2 after the treatment solution composition of the present invention is transparentized and the whole organ is directly immunolabeled with AFP and Survivin.
  • FIG. 3 is a two-photon microscope image of the brain of an adult mouse in Experiment 3 after the treatment solution kit of the present invention is transparentized and the whole tissue is indirectly immunolabeled with mTOR in the brain.
  • FIG. 4 is a two-photon microscope image of the adult rat brain in experiment 4 after the treatment solution kit of the present invention is transparentized and the whole tissue is indirectly immunolabeled with NeuN in the brain.
  • FIG. 5 is a two-photon microscope image of the spinal cord of an adult mouse in Experiment 5 after being transparentized with the treatment solution kit of the present invention and indirectly immunolabeled ⁇ -III tubulin in the spinal cord with the whole tissue.
  • FIG. 6 is a two-photon microscope image of the femoral head of the adult mouse in experiment 6 after the treatment solution kit of the present invention is transparentized and the whole tissue is indirectly immunolabeled with II-collagen in articular cartilage.
  • the invention provides a treatment liquid for making whole organs transparent, comprising sugars, urea, and nonionic surfactants.
  • the sugar is selected as a monosaccharide with a higher refractive index. If the sugar (RI is 1.48), it can match the refractive index inside the biological sample and make the tissue more transparent. However, the maximum RI of a mixture of saccharides as a solution cannot exceed 1.48, which is not enough to match the refractive index of each component in the tissue. Therefore, it is necessary to fully hydrate some solid components in the tissue or remove some tissue components. Such as cell membrane lipids. Urea breaks the non-covalent bond interactions and causes the protein to undergo reversible denaturation.
  • cell membranes play a very important role in tissue transparency and fluorescent labeling technology, and can selectively induce cell membranes to form pores in situ to achieve tissue transparency.
  • the treatment liquid of the present invention contains saccharides with a mass and volume fraction of 0 to 60%, such as sugar; urea with a mass and volume fraction of 20 to 30%; and a nonionic surfactant with a volume fraction of 5 to 30%, such as triton X-100, Tween 60 and so on.
  • the invention also provides a mixed liquid, which comprises gradient liquids of different concentrations to form a treatment liquid combination, preferably composed of several treatment liquids.
  • a mixed liquid which comprises gradient liquids of different concentrations to form a treatment liquid combination, preferably composed of several treatment liquids.
  • sugars and urea are the key variables.
  • the sugar concentration increases, the amount of urea dissolved decreases.
  • the fructose concentration is constant, the higher the urea concentration, the more obvious the swelling of the tissue; when the urea concentration is constant, the higher the fructose concentration, the more obvious the tissue shrinkage.
  • the concentrations of sugars and urea change in the same direction.
  • the primary antibody and the secondary antibody only need to be in a ratio of 1:10 to 1: 500 (the specific ratio varies depending on the type of antibody and manufacturer) It can be dissolved in a transparent treatment solution, and the obtained treatment solution or mixed solution can be used to soak biological tissues and organs.
  • Biological tissues and organs include mouse brain, rat brain, mouse spinal cord, tumor-bearing liver and articular cartilage. Immersion can be performed at 37 ° C.
  • Specimen preparation adult C57BL mice, Thy1-gcams3-GFP transgenic mice or tumor-bearing nude mice planted in situ in the liver of HePG2 cells, weighing 20-22 g, with an excess of 3% phenobarbital sodium (2ml / kg)
  • the brain, spinal cord, femur, and tumor-bearing liver were decapitated and placed in 4% PFA in a refrigerator at 4 ° C overnight and fixed again.
  • the femur was immersed in a 37 ° C water bath for 1 week in the EDTA decalcifying solution to decalcify.
  • the above-mentioned clearing treatment liquid is used to dilute and mix the fluorescently labeled primary antibody, primary antibody and fluorescently labeled secondary antibody at a concentration of 5-200ug / ml,
  • the prepared secondary antibody should be wrapped with tin foil to protect it from light and stored for future use.
  • the mixed solution of the present invention and the mixed solution prepared by the fluorescent antibody should be wrapped with tin foil to avoid light.
  • a rabbit-derived mTOR primary antibody molecular weight of 289 kD, 50 ug / ml, Cell Signal Technology
  • mice anti-rabbit mTOR secondary antibody 25ug / ml, Abcam
  • the transparent treatment solution of the present invention in a composition prepared from light, a constant temperature water bath at 37 ° C, and immerse with the treatment solution of the present invention after 1-2 days Overnight and stored in the treatment solution of the present invention until two-photon fluorescence imaging.
  • a composition formulated with a fluorescent antibody should be wrapped in tin foil to protect from light.
  • NeuN indirect immunofluorescence of 1mm brain slices of rats take rat brains, cut them into 1mm thick brain slices with rat brain matrix, rinse with 0.01M PBS, 3 times ⁇ 5min, and then place them in 5ml centrifuge tubes Inject a rabbit-derived NeuN primary antibody (molecular weight of 55 kD, 100 ug / ml, Cell Signal Technology) and a composition prepared by the transparent treatment solution of the present invention in a constant temperature water bath at 37 ° C. After the sample is transparent (1-3d), use The treatment liquid of the present invention is immersed overnight.
  • a rabbit-derived NeuN primary antibody molecular weight of 55 kD, 100 ug / ml, Cell Signal Technology
  • mice anti-rabbit NeuN secondary antibody 25ug / ml, Abcam
  • a composition prepared by the transparent treatment solution of the present invention protect it from light, keep a constant temperature water bath at 37 ° C, and immerse with the treatment solution of the present invention after 1-2 days. Overnight and stored in the treatment solution of the present invention until two-photon fluorescence imaging.
  • a composition formulated with a fluorescent antibody should be wrapped in tin foil to protect from light.
  • ⁇ -IIITubulin indirect immunofluorescence of mouse spinal cord Take the brain and spinal cord of the mouse, carefully remove the coating on the surface of the spinal cord under a stereomicroscope, rinse with 0.01 M PBS, 3 times ⁇ 5 min, and then place it in a 5 ml centrifuge tube.
  • Mouse-derived ⁇ -III Tubulin primary antibody molecular weight: 55kD, 50ug / ml, Abcam
  • the transparent treatment solution of the present invention prepared in a constant temperature water bath at 37 ° C. After the sample is transparent (1-3d), use this The treatment solution was immersed overnight.
  • a goat anti-mouse secondary antibody IgG 40ug / ml, Abcam
  • a transparent treatment solution of the present invention in a composition prepared from light, a constant temperature water bath at 37 ° C, and immerse with the treatment solution of the present invention after 1-2 days. Overnight and stored in the treatment solution of the present invention until two-photon fluorescence imaging.
  • a composition formulated with a fluorescent antibody should be wrapped in tin foil to protect from light.
  • Mouse knee joint II-collagen indirect immunofluorescence Take the femur of the hind limb of the mouse, carefully remove the muscles and coatings on the surface of the femoral head under a stereomicroscope, rinse with 0.01M PBS, 3 times ⁇ 5min, and then place in a 50ml centrifuge tube , Inject EDTA decalcification solution, 37 °C constant temperature water bath for 1 week. After the bones have softened, put them into a composition composed of a mouse-derived II-collagen primary antibody and the transparent treatment solution of the present invention, in a constant temperature water bath at 37 ° C, and after the sample is transparent (3-4d), use this The treatment solution was immersed overnight.
  • compositions prepared by goat anti-mouse secondary antibody IgG (40ug / ml, Abcam) and the transparent treatment solution of the present invention protect it from light, keep in a constant temperature water bath at 37 ° C, and immerse with the treatment solution of the present invention It was washed overnight and stored in the treatment solution of the present invention until two-photon fluorescence imaging.
  • a composition formulated with a fluorescent antibody (secondary antibody) should be wrapped in tin foil to protect from light.
  • FIG. 1 is a two-photon microscope image of a brain specimen of a Thy1-gcamp3-GFP transgenic mouse in Experiment 1 after the whole brain is directly immunofluorescent after the treatment solution of the embodiment of the present invention.
  • the treatment liquid composition of the present invention is also compatible with the fluorescent protein GFP, and allows the entry of macromolecular antibody substances, and simultaneously realizes the transparent treatment of the whole organ and the direct immunofluorescence.
  • the arrow on the left shows the Thy1-gcamp3-GFP labeled neuron
  • the arrow on the middle shows the neuron labeled with anti-GFP antibody
  • the right shows the combination of the first two
  • the arrow shows Thy1-gcamp3-GFP.
  • the coincidence of neurons with anti-GFP antibody-labeled neurons indicates that anti-GFP antibodies can enter tissues and specifically bind to GFP.
  • FIG. 2 is a two-photon microscope image of the mouse tumor-bearing liver specimen in Experiment 2 after the transparent treatment of the present invention and direct immunological labeling of AFP and Survivin to the whole organ.
  • Immunofluorescence pictures of whole liver AFP Bottom: Immunofluorescence pictures of whole liver Survivin. It can be seen that after the treatment solution of this embodiment is processed, the whole liver tissue can be simultaneously transparentized and immunofluorescently labeled.
  • the arrow in the left picture shows the cells (cytoplasm) labeled with anti-AFP antibodies carrying Alexa-488, the middle picture shows the nucleus DAPI staining, and the right picture is the combination of the first two pictures.
  • the site of the cell is complementary to the nucleus.
  • the anti-AFP antibody can enter the whole liver tissue and is specifically expressed in the cytoplasm.
  • FIG. 3 is a two-photon microscope image of the adult mouse brain (1 mm brain slice) after being transparentized with the transparent treatment solution of the present invention and indirectly immunolabeled mTOR in the brain in the whole tissue in Experiment 3.
  • FIG. The arrows in the figure show cells labeled with anti-mTOR antibodies. It can be seen that the treatment liquid of the present invention allows the primary antibody and the secondary antibody to enter and specifically bind, and simultaneously realizes the transparent treatment of mouse brain tissue and indirect immunofluorescence.
  • FIG. 4 is a two-photon microscope image of the adult rat brain (1 mm brain slice) treated with the transparent treatment solution of the present invention and the whole tissue is indirectly immunolabeled with NeuN in the brain in Experiment 4.
  • FIG. Arrows in the figure show neuron cells labeled with anti-NeuN antibodies. It can be seen that the treatment solution of the present invention allows the primary antibody and the secondary antibody to enter and specifically bind, and simultaneously realizes the transparent treatment of the brain tissue of the rat and the indirect immunofluorescence.
  • FIG. 5 is a two-photon microscope image of the spinal cord of an adult mouse in Experiment 5 after being transparentized with the transparent treatment solution of the present invention and indirectly immunolabeled ⁇ -III-Tubulin in the spinal cord with the whole tissue.
  • the striped structures shown by arrows in the figure are nerve fibers labeled with anti- ⁇ -III-Tubulin antibody. It can be seen that the treatment liquid of the present invention allows the primary antibody and the secondary antibody to enter and specifically bind, and simultaneously realizes the transparent treatment of mouse spinal cord organs and indirect immunofluorescence.
  • FIG. 6 is a two-photon microscope image of the femoral head of the adult mouse in experiment 6 after the treatment solution kit of the present invention is transparentized and the whole tissue is indirectly immunolabeled with II-collagen in articular cartilage.
  • the arrows in the figure show the type II collagen labeled with anti-II-collagen antibody, which is located on the articular surface or in the cytoplasm of chondrocytes. It can be seen that the treatment liquid of the present invention allows the primary and secondary antibodies to enter and specifically bind, and simultaneously realizes the transparent treatment and indirect immunofluorescence of the articular surface of the femoral head and the entire cartilage of the mouse.
  • the treatment solution used in the present invention is a 37 ° C water bath soaking sample at a constant temperature, without complicated pretreatment and processing procedures, and simple operation;
  • the present invention can make organs of different animals (rats, mice, etc.) and different organs (brain, spinal cord, articular cartilage, tumor-bearing liver, etc.) transparent and immunologically labeled;
  • the treatment solution used in the present invention can be formulated with a fluorescently labeled primary antibody, a primary antibody, and a fluorescently labeled secondary antibody to form an overall immunofluorescent solution, which can achieve the purpose of simultaneous transparency and immunofluorescent labeling at the level of the entire organ (see Figure 1- 6).

Abstract

A treating liquid composition, a treating liquid kit, and a biological organ transparentizing and immune labeling method. The treating liquid composition contains a transparent treating liquid and a fluorescently-labeled antibody having a volume ratio of 1:10 to 1:500 to the transparent treating liquid, wherein the transparent treating liquid contains: saccharides, carbamide, and a non-ionic surfactant. According to the method, while the whole organs such as brain, spinal cord, cartilago articularis, and tumor-burdened viscus are transparentized, biological macromolecules such as antibodies are allowed to enter organs, so as to achieve direct or indirect immune labeling of the whole organs on a scale.

Description

处理液组合物、试剂盒和生物器官透明化同时进行免疫标记的方法Treatment liquid composition, kit and biological organ transparent method and immunolabeling method 技术领域Technical field
本发明涉及生物组织整体器官透明化和免疫标记的方法，特别是涉及用于将脑、脊髓、关节软骨、脏器等整体器官在透明化的同时直接或间接免疫荧光标记的处理液和方法。The invention relates to a method for transparentizing and immunolabeling a whole organ of a biological tissue, in particular to a treatment solution and a method for directly or indirectly immunofluorescently marking a whole organ such as brain, spinal cord, articular cartilage, and organs while transparentizing.
背景技术Background technique
组织透明方法可以实现组织无损情况下的大尺度高分辨检测与组织形态的三维结构重建。相对于传统的组织切片与染色方法具有显著性的进步。目前开发的所有组织透明方法中，只有部分组织透明方案允许整体器官免疫标记，如iDisco、Clarity、Cubic、ACT-presto等。Clarity和Cubic均去除了细胞膜，允许大分子进入，但是过程繁琐，需时近1-2周；iDisco使用的是有机溶剂，荧光兼容性较差，组织变硬变脆，且刺激性气味对人体有害；ACT-presto借助压力，需要专门设备，且免疫标记前需要繁杂的前处理过程。The method of tissue transparency can realize large-scale high-resolution detection and three-dimensional structure reconstruction of tissue morphology in the case of non-destructive tissue. Compared with traditional tissue sectioning and staining methods, it has a significant improvement. Of all the tissue transparency methods currently developed, only part of the tissue transparency scheme allows for overall organ immunolabeling, such as iDisco, Clarity, Cubic, ACT-presto, etc. Clarity and Cubic both remove the cell membrane and allow macromolecules to enter, but the process is tedious and takes nearly 1-2 weeks; iDisco uses organic solvents, has poor fluorescence compatibility, tissues become hard and brittle, and the pungent odor is harmful to the human body Harmful; ACT-presto relies on pressure, requires special equipment, and requires a complicated pre-treatment process before immunolabeling.
发明内容Summary of the Invention
本发明旨在提供一种操作简单、高效的整体器官或组织透明化同时免疫标记的技术方案。The invention aims to provide a simple and efficient technical solution for the whole organ or tissue to be transparent and immunolabeled.
为了实现上述目的，作为本发明的一个方面，本发明提供了一种处理液组合物，包含：In order to achieve the above object, as one aspect of the present invention, the present invention provides a treatment liquid composition including:
透明化处理液；和Clearing treatment fluid; and
荧光标记抗体，所述荧光标记抗体与所述透明化处理液的体积比为1:10至1:500，A fluorescently labeled antibody, and a volume ratio of the fluorescently labeled antibody to the clearing treatment solution is 1:10 to 1: 500,
其中，所述透明化处理液包含：Wherein, the transparent treatment liquid includes:
糖类；carbohydrate;
尿素；Urea
非离子表及面活性剂，所述非离子表面活性剂包括体积比为5:3至1:5的曲Non-ionic surface and surfactant, the non-ionic surfactant includes koji with a volume ratio of 5: 3 to 1: 5
拉通和吐温混合物。Pull through and tween the mixture.
在某些实施方案中，糖类为果糖或蔗糖中的至少一种。In certain embodiments, the sugar is at least one of fructose or sucrose.
在某些实施方案中，曲拉通包括曲拉通X-100和曲拉通X-114。In some embodiments, Triton includes Triton X-100 and Triton X-114.
在某些实施方案中，吐温包括吐温20，吐温40和吐温60。In certain embodiments, Tween includes Tween 20, Tween 40 and Tween 60.
在某些实施方案中，每100ml透明化处理液包含：20-30g的尿素，10-15ml的非离子表面活性剂，40-60g的糖类，0-20ml的氨类，0-2ml的抗氧化剂，余量为水。In some embodiments, each 100 ml of the clearing treatment liquid contains: 20-30 g of urea, 10-15 ml of non-ionic surfactant, 40-60 g of sugars, 0-20 ml of ammonia, 0-2 ml of anti-bacterial Oxidant, the balance is water.
在某些实施方案中，透明化处理液为一系列处理液，其中随着糖类含量的增加，尿素含量也增加，或者随着糖类含量的减少，尿素含量也减少。In some embodiments, the clearing treatment liquid is a series of treatment liquids, wherein as the sugar content increases, the urea content also increases, or as the sugar content decreases, the urea content also decreases.
在某些实施方案中，抗体包括以下抗体中的至少一种：anti-AFP抗体，分子量约65kD，优选使用浓度范围为5-25μg/ml；anti-Survivin抗体，分子量约16kD，优选使用浓度范围为5-25μg/ml；和anti-GFP抗体，分子量约27kD，优选使用浓度范围为10-100ug/ml。In certain embodiments, the antibody includes at least one of the following: anti-AFP antibody, with a molecular weight of about 65 kD, preferably in a concentration range of 5-25 μg / ml; anti-Survivin antibody, with a molecular weight of about 16 kD, preferably in a concentration range It is 5-25 μg / ml; and anti-GFP antibody, the molecular weight is about 27kD, and the concentration range is preferably 10-100ug / ml.
作为本发明的另一个方面，本发明还提供一种处理液试剂盒，其包含：As another aspect of the present invention, the present invention also provides a treatment solution kit, comprising:
第一部分，包括：透明化处理液和按1:10至1:500体积比与透明化处理液混合的一抗；和The first part includes: a transparent treatment liquid and a primary antibody mixed with the transparent treatment liquid in a volume ratio of 1:10 to 1: 500; and
第二部分，包括：透明化处理液和按1:10至1:500体积比与透明化处理液混合的带荧光标记的二抗，The second part includes: a transparent solution and a fluorescently labeled secondary antibody mixed with the transparent solution in a volume ratio of 1:10 to 1: 500,
其中的透明化处理液为上述的透明化处理液。The clearing treatment liquid is the aforementioned clearing treatment liquid.
在某些实施方案中，处理液试剂盒中的一抗包括：In certain embodiments, the primary antibodies in the treatment solution kit include:
anti-mTOR一抗(抗哺乳动物雷帕霉素靶蛋白)，分子量约289kD，优选使用浓度范围为10-50μg/ml；anti-mTOR primary antibody (anti-mammalian rapamycin target protein), with a molecular weight of about 289 kD, preferably in a concentration range of 10-50 μg / ml;
anti-β-Ⅲ Tubulin一抗(抗β-Ⅲ型微管蛋白抗体)，分子量约55kD，优选使用浓度范围为10-50μg/ml；anti-β-Ⅲ Tubulin primary antibody (anti-β-Ⅲ tubulin antibody), molecular weight of about 55kD, preferably in a concentration range of 10-50 μg / ml;
anti-NeuN一抗(抗神经元特异性核蛋白抗体)，分子量46-55kD，优选使用浓度范围为10-200μg/ml；或anti-NeuN primary antibody (anti-neuron-specific nuclear protein antibody), molecular weight 46-55kD, preferably in a concentration range of 10-200 μg / ml; or
anti-Ⅱ collagen一抗(抗Ⅱ型胶原蛋白抗体)，分子量约142kD，小鼠来源，优选使用浓度范围为10-200μg/ml，anti-Ⅱcollagen primary antibody (anti-type II collagen antibody), molecular weight about 142kD, mouse origin, preferably used in the concentration range of 10-200μg / ml,
所述的带荧光标记的二抗是携带荧光标记的、能够与一抗特异性结合(抗小鼠或抗兔)的免疫球蛋白(IgG)。The fluorescently labeled secondary antibody is a fluorescently labeled immunoglobulin (IgG) capable of specifically binding to the primary antibody (anti-mouse or anti-rabbit).
作为本发明的另一个方面，本发明还提供一种生物器官透明化同时进行免疫标记的方法，包括用上述的处理液组合物浸泡生物组织器官；优选地，浸泡在37℃恒温进行1-7天。As another aspect of the present invention, the present invention also provides a method for transparentizing biological organs and performing immunolabeling at the same time, including soaking biological tissues and organs with the above-mentioned treatment liquid composition; preferably, soaking at 37 ° C for 1-7 day.
在某些实施方案中，将生物组织器官浸泡在处理液组合物中，待组织透明化后，用不含抗体的透明化处理液清洗以除去未结合的抗体。In certain embodiments, the biological tissue organs are immersed in the treatment solution composition, and after the tissue is cleared, the tissue is washed with an antibody-free clearing treatment solution to remove unbound antibodies.
作为本发明的另一个方面，本发明还提供一种生物器官透明化同时进行免疫标记的方法，包括用上述的处理液试剂盒处理生物组织器官，其中As another aspect of the present invention, the present invention also provides a method for transparentizing biological organs and simultaneously performing immunolabeling, which includes treating biological tissues and organs with the above-mentioned treatment solution kit, wherein
先用第一部分浸泡生物组织器官，优选地，浸泡在37℃恒温进行1-4天；First immerse the biological tissues and organs with the first part, preferably, soak at 37 ° C for 1-4 days;
然后，用第二部分浸泡生物组织器官，优选地，浸泡在37℃恒温进行1-3天。Then, the second part is used to soak the biological tissues and organs, preferably at 37 ° C for 1-3 days.
在某些实施方案中，先将生物组织器官浸泡在用透明化处理液稀释的一抗溶液中，待组织透明化后，用透明化处理液清洗以除去未结合的一抗；然后用透明化处理液稀释的带荧光标记的二抗溶液浸泡生物组织器官，约1-3天后，用不含抗体的透明化处理液清洗以除去未结合的二抗。In some embodiments, biological tissues and organs are first immersed in a primary antibody solution diluted with a clearing treatment solution. After the tissue is cleared, the clearing treatment solution is used to remove unbound primary antibodies; Soak biological tissues and organs with the fluorescently labeled secondary antibody solution diluted in the treatment solution. After about 1-3 days, wash with a clearing treatment solution without antibodies to remove unbound secondary antibodies.
在某些实施方案中，所述生物组织器官包括脑、脊髓、关节软骨以及脏器(包括荷瘤脏器)。In certain embodiments, the biological tissue organs include brain, spinal cord, articular cartilage, and organs (including tumor-bearing organs).
在某些实施方案中，生物组织器官包括小鼠脑、大鼠脑、小鼠脊髓及关节软骨等组织器官，依据组织块大小，浸泡在37℃恒温进行1-7天。In some embodiments, the biological tissues and organs include tissues such as mouse brain, rat brain, mouse spinal cord, and articular cartilage. Depending on the size of the tissue mass, they are immersed in a constant temperature at 37 ° C for 1-7 days.
本发明提供了一种新的整体器官尺度上组织直接或间接免疫荧光的方法。该方法在组织透明化的同时实现整体器官尺度上的免疫荧光标记。The present invention provides a new method for direct or indirect immunofluorescence of tissues at the overall organ scale. This method realizes the immunofluorescence labeling on the whole organ scale while the tissue is transparent.
根据本发明，整个操作过程简单，不需要特殊实验装置；本发明方法的免疫荧光效果确定，而且可以以较快速度进行。According to the present invention, the entire operation process is simple, and no special experimental device is required; the immunofluorescence effect of the method of the present invention is determined, and it can be performed at a relatively fast speed.
附图说明：Brief description of the drawings:
图1为试验1中小鼠脑标本经本发明的处理液组合物整脑直接免疫荧光后的双光子显微镜图像。FIG. 1 is a two-photon microscope image of a mouse brain specimen in Experiment 1 after direct immunofluorescence of whole brain of the treatment solution composition of the present invention.
图2为试验2中小鼠荷瘤肝标本经本发明的处理液组合物透明化处理并整体器官直接免疫标记AFP及Survivin后的双光子显微镜图像。FIG. 2 is a two-photon microscope image of a mouse tumor-bearing liver specimen in Test 2 after the treatment solution composition of the present invention is transparentized and the whole organ is directly immunolabeled with AFP and Survivin.
图3为试验3中成年小鼠脑经本发明处理液试剂盒透明化处理并整体组织间接免疫标记脑中mTOR后的双光子显微镜图像。FIG. 3 is a two-photon microscope image of the brain of an adult mouse in Experiment 3 after the treatment solution kit of the present invention is transparentized and the whole tissue is indirectly immunolabeled with mTOR in the brain.
图4为试验4中成年大鼠脑经本发明处理液试剂盒透明化处理并整体组织间接免疫标记脑中NeuN后的双光子显微镜图像。FIG. 4 is a two-photon microscope image of the adult rat brain in experiment 4 after the treatment solution kit of the present invention is transparentized and the whole tissue is indirectly immunolabeled with NeuN in the brain.
图5为试验5中成年小鼠脊髓经本发明处理液试剂盒透明化处理并整体组织间接免疫标记脊髓中β-Ⅲ tubulin后的双光子显微镜图像。FIG. 5 is a two-photon microscope image of the spinal cord of an adult mouse in Experiment 5 after being transparentized with the treatment solution kit of the present invention and indirectly immunolabeled β-III tubulin in the spinal cord with the whole tissue.
图6为试验6中成年小鼠股骨头经本发明处理液试剂盒透明化处理并整体组织间接免疫标记关节软骨中的Ⅱ-collagen后的双光子显微镜图像。FIG. 6 is a two-photon microscope image of the femoral head of the adult mouse in experiment 6 after the treatment solution kit of the present invention is transparentized and the whole tissue is indirectly immunolabeled with II-collagen in articular cartilage.
具体实施方式detailed description
本发明提供了一种使完整器官透明的处理液，包含糖类、尿素、非离子表面活性剂。糖类选择为具有较高折射率的单糖例如果糖(RI为1.48)，可以实现生物样本内 部折光率的匹配，使组织更透明。但是以糖类为溶液的混合物，最高RI不能超过1.48，这对于实现组织内部各成分折光率的匹配是不够的，因此，需要将组织中某些固态成分充分水化，或者去除一些组织成分，如细胞膜脂质。尿素打破非共价键的相互作用，使蛋白质发生可逆性变性，它的加入增加样品中膜蛋白的溶解性，使其充分水化，降低其折光率。但是，仅采用糖类和尿素，生物组织器官的透明化需要很长的时间，而且存在与免疫荧光标记技术不兼容的问题。本发明的发明人发现细胞膜在组织透明化和荧光标记技术中起着非常重要的作用，可以选择性的诱导细胞膜在原位形成孔隙从而实现组织透明。其孔隙率和大小显著影响透明化的速度和效果以及大分子物质如抗体进入细胞；通过采用特定的非离子性表面活性剂(选自曲拉通和吐温两种的组合)，可以使得细胞膜具有合适的孔隙率和大小，从而实现快速透明化并与免疫标记技术兼容。但是，乳化作用较强的疏水长链非离子表面活性剂或表面活性剂过量也会破坏细胞蛋白结构，从而影响荧光及免疫染色。经过反复试验，我们发现仅用曲拉通X-100或者曲拉通X-100含量过高时，组织结构会被破坏，而仅用吐温60时，透明时间长，透明效果不佳。只有在同时应用曲拉通X-100及吐温60时，且在合适的比例时(5:3-1:5)，可在较短的时间内达到较好的透明效果。The invention provides a treatment liquid for making whole organs transparent, comprising sugars, urea, and nonionic surfactants. The sugar is selected as a monosaccharide with a higher refractive index. If the sugar (RI is 1.48), it can match the refractive index inside the biological sample and make the tissue more transparent. However, the maximum RI of a mixture of saccharides as a solution cannot exceed 1.48, which is not enough to match the refractive index of each component in the tissue. Therefore, it is necessary to fully hydrate some solid components in the tissue or remove some tissue components. Such as cell membrane lipids. Urea breaks the non-covalent bond interactions and causes the protein to undergo reversible denaturation. Its addition increases the solubility of membrane proteins in the sample, makes it fully hydrated, and reduces its refractive index. However, using only sugars and urea, the transparency of biological tissues and organs takes a long time, and there is a problem of incompatibility with immunofluorescence labeling technology. The inventors of the present invention have found that cell membranes play a very important role in tissue transparency and fluorescent labeling technology, and can selectively induce cell membranes to form pores in situ to achieve tissue transparency. Its porosity and size significantly affect the speed and effect of transparency and the entry of macromolecular substances such as antibodies into cells; by using specific non-ionic surfactants (selected from the combination of Triton and Tween), the cell membrane can be made Have the right porosity and size for fast transparency and compatibility with immunolabeling technology. However, hydrophobic long-chain nonionic surfactants with strong emulsification effects or excessive surfactants can also damage the cellular protein structure, which affects fluorescence and immunostaining. After repeated experiments, we found that when only Triton X-100 or Triton X-100 content is too high, the tissue structure will be destroyed, and when only Tween 60 is used, the transparency time is long and the transparency effect is not good. Only when Triton X-100 and Tween 60 are applied at the same time, and at the appropriate ratio (5: 3-1: 5), a better transparency effect can be achieved in a short period of time.
本发明的处理液包含质量体积分数为0～60％的糖类，例如果糖；质量体积分数为20～30％的尿素；体积分数为5-30％的非离子表面活性剂，如曲拉通X-100，吐温60等。The treatment liquid of the present invention contains saccharides with a mass and volume fraction of 0 to 60%, such as sugar; urea with a mass and volume fraction of 20 to 30%; and a nonionic surfactant with a volume fraction of 5 to 30%, such as triton X-100, Tween 60 and so on.
本发明还提供一种混合液，包括不同浓度梯度物质组成一个处理液组合，优选由几种处理液组成。对于该处理液组合，糖类和尿素是关键变量。糖类浓度升高时，尿素溶解量下降。但是经过多次试验，我们发现在组织透明液中，当果糖浓度恒定时，尿素浓度越高，组织膨胀越明显；当尿素浓度恒定时，果糖浓度越高，组织皱缩越明显。本发明中，糖类和尿素的浓度同方向变化。为防止组织变形、保证折光率的匹配及免疫染色，选择糖类为0-60％，尿素为20-30％，且两者同向变化，以达到一个既能水化膜蛋白、匹配折光率，又能保证组织不变形，并允许免疫标记的平衡。The invention also provides a mixed liquid, which comprises gradient liquids of different concentrations to form a treatment liquid combination, preferably composed of several treatment liquids. For this treatment solution combination, sugars and urea are the key variables. When the sugar concentration increases, the amount of urea dissolved decreases. However, after several experiments, we found that when the fructose concentration is constant, the higher the urea concentration, the more obvious the swelling of the tissue; when the urea concentration is constant, the higher the fructose concentration, the more obvious the tissue shrinkage. In the present invention, the concentrations of sugars and urea change in the same direction. In order to prevent tissue deformation, ensure the matching of refractive index and immunostaining, choose 0-60% sugars and 20-30% urea, and both of them change in the same direction to achieve a hydration membrane protein that matches the refractive index It also guarantees that the tissue does not deform and allows the balance of the immune markers.
在具体实施方案的选择中，要综合考虑外源性生物大分子的通透性，组织的透明度，还有操作的简便性、速度、安全性、经济等各个方面，有时不得不在保证主要目的的前提下，对其他方面的特性进行让步。In the choice of specific implementation schemes, we must comprehensively consider the permeability of exogenous biological macromolecules, the transparency of the organization, as well as the simplicity, speed, safety, and economy of the operation. Sometimes, we have to ensure that the main purpose is On the premise, make concessions to other aspects.
在本发明中，作为使生物组织器官透明化同时进行免疫标记的方法，只需将一抗及二抗以1:10-1:500的比例(具体的比例因抗体种类及厂家不同而不同)溶于透明处理液中，并且用所得的处理液或混合液浸泡生物组织器官即可。In the present invention, as a method for making biological tissues and organs transparent while performing immunolabeling, the primary antibody and the secondary antibody only need to be in a ratio of 1:10 to 1: 500 (the specific ratio varies depending on the type of antibody and manufacturer) It can be dissolved in a transparent treatment solution, and the obtained treatment solution or mixed solution can be used to soak biological tissues and organs.
生物组织器官包括小鼠脑、大鼠脑、小鼠脊髓、荷瘤肝及关节软骨等组织器官等。浸泡可以在37℃恒温进行。Biological tissues and organs include mouse brain, rat brain, mouse spinal cord, tumor-bearing liver and articular cartilage. Immersion can be performed at 37 ° C.
下面结合具体实施例，并参照附图，对本发明作进一步的详细说明。The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings.
(1)标本制备：成年C57BL小鼠，Thy1-gcams3-GFP转基因小鼠或HePG2细胞肝脏原位种植的荷瘤裸鼠，体重20-22g，过量3％苯巴比妥钠(2ml/kg)腹腔注射麻醉，用4％PFA经心脏灌注后，断头取脑，脊髓，股骨及荷瘤肝，置于4％PFA中4℃冰箱中过夜，再次固定。其中，股骨置于EDTA脱钙液中37℃水浴浸泡1周以脱去钙质。(1) Specimen preparation: adult C57BL mice, Thy1-gcams3-GFP transgenic mice or tumor-bearing nude mice planted in situ in the liver of HePG2 cells, weighing 20-22 g, with an excess of 3% phenobarbital sodium (2ml / kg) After intraperitoneal anesthesia, after perfusion with 4% PFA through the heart, the brain, spinal cord, femur, and tumor-bearing liver were decapitated and placed in 4% PFA in a refrigerator at 4 ° C overnight and fixed again. Among them, the femur was immersed in a 37 ° C water bath for 1 week in the EDTA decalcifying solution to decalcify.
成年SD大鼠，体重约200-250g左右，过量3％苯巴比妥钠(2ml/kg)腹腔注射麻醉，用4％PFA经心脏灌注后，断头取脑，置于4％PFA中4℃冰箱中过夜，再次固定。Adult SD rats, weighing about 200-250 g, were anesthetized by intraperitoneal injection of 3% phenobarbital sodium (2ml / kg), and after perfusion with 4% PFA through the heart, the brain was decapitated and placed in 4% PFA. 4 Overnight in refrigerator at ℃ and fixed again.
(2)透明化处理液的配制：称取52g果糖于200ml的烧杯,置于60℃水浴中溶解，再加入24g尿素，5ml曲拉通X-100，6ml吐温60，去离子水定容至100ml，室温避光保存。(2) Preparation of transparent treatment solution: Weigh 52g of fructose in a 200ml beaker, dissolve it in a water bath at 60 ° C, then add 24g of urea, 5ml of Triton X-100, 6ml of Tween 60, and make up with deionized water To 100ml, store at room temperature in the dark.
(3)荧光抗体处理液组合物的配制：用上述配制好的透明化处理液将荧光标记的一抗，一抗及荧光标记的二抗以5-200ug/ml的浓度进行稀释，混匀，配制的二抗需用锡纸包裹以避光存放，待用。(3) Preparation of the fluorescent antibody treatment liquid composition: the above-mentioned clearing treatment liquid is used to dilute and mix the fluorescently labeled primary antibody, primary antibody and fluorescently labeled secondary antibody at a concentration of 5-200ug / ml, The prepared secondary antibody should be wrapped with tin foil to protect it from light and stored for future use.
(4)整体组织尺度上的直接免疫荧光标记：(4) Direct immunofluorescence labeling on the overall tissue scale:
试验1Test 1
整脑免疫荧光——取小鼠脑，用1×PBS冲洗，3次×5min，然后置于5ml装有共轭594荧光素的抗GFP抗体(分子量27kD，50ug/ml，Thermo Fisher)透明溶液的离心管中，37℃恒温水浴，待样本透亮后，用不含荧光抗体的本发明混合液冲洗2h，并保存在本发明的混合液中直至双光子荧光成像。在整个过程中，装有转基因小鼠脑的离心管应包裹锡纸以避光。Whole brain immunofluorescence-take the mouse brain, wash it with 1 × PBS, 3 times × 5min, and then place it in 5ml of anti-GFP antibody (molecular weight 27kD, 50ug / ml, Thermo Fisher) transparent solution containing 594 fluorescein In a centrifuge tube, a constant temperature water bath at 37 ° C, after the sample is transparent, rinse with the mixed solution of the present invention containing no fluorescent antibody for 2 h, and store in the mixed solution of the present invention until two-photon fluorescence imaging. Throughout the process, centrifuge tubes containing the brains of transgenic mice should be wrapped in tin foil to protect them from light.
试验2Test 2
整肝免疫荧光——取荷瘤肝，用用1×PBS冲洗，3次×5min，然后置于5ml装有共轭488荧光素的抗AFP抗体(分子量70kD，25ug/ml，Abcam)及抗Survivin抗体(分子量16KD，25ug/ml，Cell Signal Technology)透明溶液的离心管中，37℃恒温水浴，待样本透亮后(1-3d)，用本发明的混合液冲洗2h，摇床轻摇；然后放入用本发明的混合液配置的DAPI核染液(0.2mg/ml,索莱宝)中,常温，1d后，用本发明的混合液冲洗2h，摇床轻摇；然后，保存在本发明的混合液中直至双光子荧光成像。在整个过程中，本发明的混合液与荧光抗体配制的混合液组合应包裹锡纸以避光。Immunofluorescence of whole liver——Tumor-bearing liver was taken, washed with 1 × PBS, 3 times × 5min, and then placed in 5ml of anti-AFP antibody (molecular weight 70kD, 25ug / ml, Abcam) and anti-AFP Survivin antibody (molecular weight 16KD, 25ug / ml, Cell Signal Technology) in a centrifuge tube of a transparent solution, a constant temperature water bath at 37 ° C, after the sample is transparent (1-3d), rinse with the mixed solution of the present invention for 2h, shake gently Then put it into the DAPI nuclear staining solution (0.2mg / ml, Solvay) configured with the mixed solution of the present invention. After 1 day at room temperature, rinse with the mixed solution of the present invention for 2h, shake it gently on the shaker; then, store it in Up to two-photon fluorescence imaging in the mixed solution of the present invention. Throughout the whole process, the mixed solution of the present invention and the mixed solution prepared by the fluorescent antibody should be wrapped with tin foil to avoid light.
(5)整体组织尺度上的间接免疫荧光标记：(5) Indirect immunofluorescence labeling on the overall tissue scale:
试验3Test 3
小鼠1mm脑片mTOR间接免疫荧光——取小鼠脑，用小鼠脑matrix切成冠状面为1mm厚的脑片，用1×PBS冲洗，3次×5min，然后置于5ml离心管中，注入兔源的mTOR第一抗体(分子量为289kD，50ug/ml，Cell Signal Technology)与本发明透明处理液配制的组合物中，37℃恒温水浴，待样本透亮后(1-3d)，用本发明处理液浸洗过夜。然后用小鼠抗兔的mTOR第二抗体(25ug/ml,Abcam)与本发明透明处理液配制的组合物中，避光，37℃恒温水浴，1-2d后，用本发明处理液浸洗过夜并保存在本发明的处理液中直至双光子荧光成像。在整个过程中，荧光抗体(二抗)配制的组合物应包裹锡纸以避光。Mouse 1mm brain slice mTOR indirect immunofluorescence-Take the mouse brain, cut it into a 1mm thick brain slice with the mouse brain matrix, rinse with 1 × PBS, 3 times × 5min, and then place it in a 5ml centrifuge tube Inject a rabbit-derived mTOR primary antibody (molecular weight of 289 kD, 50 ug / ml, Cell Signal Technology) and a composition prepared by the transparent treatment solution of the present invention in a constant temperature water bath at 37 ° C. After the sample is transparent (1-3d), use The treatment liquid of the present invention is immersed overnight. Then use a mouse anti-rabbit mTOR secondary antibody (25ug / ml, Abcam) and the transparent treatment solution of the present invention in a composition prepared from light, a constant temperature water bath at 37 ° C, and immerse with the treatment solution of the present invention after 1-2 days Overnight and stored in the treatment solution of the present invention until two-photon fluorescence imaging. Throughout the process, a composition formulated with a fluorescent antibody (secondary antibody) should be wrapped in tin foil to protect from light.
试验4Test 4
大鼠1mm脑片NeuN间接免疫荧光——取大鼠脑，用大鼠脑matrix切成冠状面为1mm厚的脑片，用0.01M PBS冲洗，3次×5min，然后置于5ml离心管中，注入兔源的NeuN第一抗体(分子量为55kD，100ug/ml，Cell Signal Technology)与本发明透明处理液配制的组合物中，37℃恒温水浴，待样本透亮后(1-3d)，用本发明处理液浸洗过夜。然后用小鼠抗兔的NeuN第二抗体(25ug/ml,Abcam)与本发明透明处理液配制的组合物中，避光，37℃恒温水浴，1-2d后，用本发明处理液浸洗过夜并保存在本发明的处理液中直至双光子荧光成像。在整个过程中，荧光抗体(二抗)配制的组合物应包裹锡纸以避光。NeuN indirect immunofluorescence of 1mm brain slices of rats—take rat brains, cut them into 1mm thick brain slices with rat brain matrix, rinse with 0.01M PBS, 3 times × 5min, and then place them in 5ml centrifuge tubes Inject a rabbit-derived NeuN primary antibody (molecular weight of 55 kD, 100 ug / ml, Cell Signal Technology) and a composition prepared by the transparent treatment solution of the present invention in a constant temperature water bath at 37 ° C. After the sample is transparent (1-3d), use The treatment liquid of the present invention is immersed overnight. Then, use a mouse anti-rabbit NeuN secondary antibody (25ug / ml, Abcam) and a composition prepared by the transparent treatment solution of the present invention, protect it from light, keep a constant temperature water bath at 37 ° C, and immerse with the treatment solution of the present invention after 1-2 days. Overnight and stored in the treatment solution of the present invention until two-photon fluorescence imaging. Throughout the process, a composition formulated with a fluorescent antibody (secondary antibody) should be wrapped in tin foil to protect from light.
试验5Test 5
小鼠脊髓β-Ⅲ Tubulin间接免疫荧光——取小鼠脑脊髓，在体式显微镜下小心剔除脊髓表面的被膜，用0.01M PBS冲洗，3次×5min，然后置于5ml离心管中，注入小鼠源的β-Ⅲ Tubulin第一抗体(分子量为55kD，50ug/ml，Abcam)与本发明透明处理液配制的组合物中，37℃恒温水浴，待样本透亮后(1-3d)，用本发明处理液浸洗过夜。然后用山羊抗小鼠的第二抗体IgG(40ug/ml,Abcam)与本发明透明处理液配制的组合物中，避光，37℃恒温水浴，1-2d后，用本发明处理液浸洗过夜并保存在本发明的处理液中直至双光子荧光成像。在整个过程中，荧光抗体(二抗)配制的组合物应包裹锡纸以避光。Β-ⅢTubulin indirect immunofluorescence of mouse spinal cord——Take the brain and spinal cord of the mouse, carefully remove the coating on the surface of the spinal cord under a stereomicroscope, rinse with 0.01 M PBS, 3 times × 5 min, and then place it in a 5 ml centrifuge tube. Mouse-derived β-Ⅲ Tubulin primary antibody (molecular weight: 55kD, 50ug / ml, Abcam) and the transparent treatment solution of the present invention, prepared in a constant temperature water bath at 37 ° C. After the sample is transparent (1-3d), use this The treatment solution was immersed overnight. Then use a goat anti-mouse secondary antibody IgG (40ug / ml, Abcam) and a transparent treatment solution of the present invention in a composition prepared from light, a constant temperature water bath at 37 ° C, and immerse with the treatment solution of the present invention after 1-2 days. Overnight and stored in the treatment solution of the present invention until two-photon fluorescence imaging. Throughout the process, a composition formulated with a fluorescent antibody (secondary antibody) should be wrapped in tin foil to protect from light.
试验6Test 6
小鼠膝关节Ⅱ-collagen间接免疫荧光——取小鼠后肢股骨，在体式显微镜下小心剔除股骨头表面的肌肉和被膜，用0.01M PBS冲洗，3次×5min，然后置于50ml离心管中，注入EDTA脱钙液，37℃恒温水浴1周。待骨骼变软后，将其放入由小鼠源的Ⅱ-collagen第一抗体与本发明透明处理液配置的组合物中，37℃恒温水浴，待样本透亮后(3-4d)，用本发明处理液浸洗过夜。然后置于山羊抗小鼠的第二抗体IgG(40ug/ml, Abcam)与本发明透明处理液配制的组合物中，避光，37℃恒温水浴，2-3d后，用本发明处理液浸洗过夜并保存在本发明的处理液中直至双光子荧光成像。在整个过程中，荧光抗体(二抗)配制的组合物应包裹锡纸以避光。Mouse knee joint Ⅱ-collagen indirect immunofluorescence——Take the femur of the hind limb of the mouse, carefully remove the muscles and coatings on the surface of the femoral head under a stereomicroscope, rinse with 0.01M PBS, 3 times × 5min, and then place in a 50ml centrifuge tube , Inject EDTA decalcification solution, 37 ℃ constant temperature water bath for 1 week. After the bones have softened, put them into a composition composed of a mouse-derived II-collagen primary antibody and the transparent treatment solution of the present invention, in a constant temperature water bath at 37 ° C, and after the sample is transparent (3-4d), use this The treatment solution was immersed overnight. Then place it in a composition prepared by goat anti-mouse secondary antibody IgG (40ug / ml, Abcam) and the transparent treatment solution of the present invention, protect it from light, keep in a constant temperature water bath at 37 ° C, and immerse with the treatment solution of the present invention It was washed overnight and stored in the treatment solution of the present invention until two-photon fluorescence imaging. Throughout the process, a composition formulated with a fluorescent antibody (secondary antibody) should be wrapped in tin foil to protect from light.
图1至图6显示了部分试验结果：Figures 1 to 6 show some test results:
图1为试验1中Thy1-gcamp3-GFP转基因小鼠脑标本经本发明实施例的处理液整脑直接免疫荧光后的双光子显微镜图像。可见本发明处理液组合物与荧光蛋白GFP也是兼容的，且允许大分子抗体物质进入，同步实现了整体器官的透明化处理及直接免疫荧光。图中，左图箭头所示为Thy1-gcamp3-GFP标记神经元，中图箭头所示为抗GFP抗体标记的神经元，右图为前两图合并，箭头所示为Thy1-gcamp3-GFP标记神经元与抗GFP抗体标记神经元的重合，表明抗GFP抗体可以进入组织与GFP特异性结合。FIG. 1 is a two-photon microscope image of a brain specimen of a Thy1-gcamp3-GFP transgenic mouse in Experiment 1 after the whole brain is directly immunofluorescent after the treatment solution of the embodiment of the present invention. It can be seen that the treatment liquid composition of the present invention is also compatible with the fluorescent protein GFP, and allows the entry of macromolecular antibody substances, and simultaneously realizes the transparent treatment of the whole organ and the direct immunofluorescence. In the figure, the arrow on the left shows the Thy1-gcamp3-GFP labeled neuron, the arrow on the middle shows the neuron labeled with anti-GFP antibody, the right shows the combination of the first two, and the arrow shows Thy1-gcamp3-GFP. The coincidence of neurons with anti-GFP antibody-labeled neurons indicates that anti-GFP antibodies can enter tissues and specifically bind to GFP.
图2为试验2中小鼠荷瘤肝标本经本发明的透明化处理并整体器官直接免疫标记AFP及Survivin后的双光子显微镜图像。上图：整肝AFP免疫荧光图片；下图：整肝Survivin免疫荧光图片。可见，经过该实施例处理液处理后，整肝组织可实现同步透明化及免疫荧光标记。上图：左图中箭头所示为携带Alexa-488的抗AFP抗体标记细胞(胞浆)，中图为细胞核DAPI染色，右图为前两图的合并，可见携带Alexa-488的抗AFP标记的细胞部位与核互补，可见抗AFP的抗体可进入整体肝组织，并特异地在胞浆中表达。下图：左图中箭头所示为携带Alexa-488的抗Survivin抗体标记细胞(胞核)，中图为细胞核DAPI染色，右图为前两图的合并。可见抗Survivin标记的细胞部位与核重叠，可见携带Alexa-488的抗Survivin可以也进入整体肝组织，并且在荷瘤肝的肿瘤细胞核中特异性表达。FIG. 2 is a two-photon microscope image of the mouse tumor-bearing liver specimen in Experiment 2 after the transparent treatment of the present invention and direct immunological labeling of AFP and Survivin to the whole organ. Above: Immunofluorescence pictures of whole liver AFP; Bottom: Immunofluorescence pictures of whole liver Survivin. It can be seen that after the treatment solution of this embodiment is processed, the whole liver tissue can be simultaneously transparentized and immunofluorescently labeled. Above: The arrow in the left picture shows the cells (cytoplasm) labeled with anti-AFP antibodies carrying Alexa-488, the middle picture shows the nucleus DAPI staining, and the right picture is the combination of the first two pictures. The site of the cell is complementary to the nucleus. It can be seen that the anti-AFP antibody can enter the whole liver tissue and is specifically expressed in the cytoplasm. Bottom: The arrow on the left shows the cells (nucleus) labeled with anti-Survivin antibody carrying Alexa-488, the middle is DAPI staining of the nucleus, and the right is the combination of the first two. It can be seen that the anti-Survivin labeled cell site overlaps with the nucleus, and it can be seen that the anti-Survivin carrying Alexa-488 can also enter the whole liver tissue, and is specifically expressed in the nucleus of tumor cells bearing tumors.
图3为试验3中成年小鼠脑(1mm脑片)经本发明透明处理液透明化处理并整体组织间接免疫标记脑中mTOR后的双光子显微镜图像。图中箭头所示为抗mTOR抗体所标记细胞。可见，本发明处理液允许一抗及二抗进入并特异性结合，同步实现了小鼠脑整体组织的透明化处理及间接免疫荧光。FIG. 3 is a two-photon microscope image of the adult mouse brain (1 mm brain slice) after being transparentized with the transparent treatment solution of the present invention and indirectly immunolabeled mTOR in the brain in the whole tissue in Experiment 3. FIG. The arrows in the figure show cells labeled with anti-mTOR antibodies. It can be seen that the treatment liquid of the present invention allows the primary antibody and the secondary antibody to enter and specifically bind, and simultaneously realizes the transparent treatment of mouse brain tissue and indirect immunofluorescence.
图4为试验4中成年大鼠脑(1mm脑片)经本发明透明处理液透明化处理并整体组织间接免疫标记脑中NeuN后的双光子显微镜图像。图中箭头所示为抗NeuN抗体标记的神经元细胞。可见本发明处理液允许一抗及二抗进入并特异性结合，同步实现了大鼠脑整体组织的透明化处理及间接免疫荧光。FIG. 4 is a two-photon microscope image of the adult rat brain (1 mm brain slice) treated with the transparent treatment solution of the present invention and the whole tissue is indirectly immunolabeled with NeuN in the brain in Experiment 4. FIG. Arrows in the figure show neuron cells labeled with anti-NeuN antibodies. It can be seen that the treatment solution of the present invention allows the primary antibody and the secondary antibody to enter and specifically bind, and simultaneously realizes the transparent treatment of the brain tissue of the rat and the indirect immunofluorescence.
图5为试验5中成年小鼠脊髓经本发明透明处理液透明化处理并整体组织间接免疫标记脊髓中β-Ⅲ-Tubulin后的双光子显微镜图像。图中箭头所示条纹状结构为抗β- Ⅲ-Tubulin抗体所标记的神经纤维。可见本发明处理液允许一抗及二抗进入并特异性结合，同步实现了小鼠脊髓整体器官的透明化处理及间接免疫荧光。FIG. 5 is a two-photon microscope image of the spinal cord of an adult mouse in Experiment 5 after being transparentized with the transparent treatment solution of the present invention and indirectly immunolabeled β-III-Tubulin in the spinal cord with the whole tissue. The striped structures shown by arrows in the figure are nerve fibers labeled with anti-β-Ⅲ-Tubulin antibody. It can be seen that the treatment liquid of the present invention allows the primary antibody and the secondary antibody to enter and specifically bind, and simultaneously realizes the transparent treatment of mouse spinal cord organs and indirect immunofluorescence.
图6为试验6中成年小鼠股骨头经本发明处理液试剂盒透明化处理并整体组织间接免疫标记关节软骨中的Ⅱ-collagen后的双光子显微镜图像。图中箭头所示为抗Ⅱ-collagen抗体所标记的Ⅱ型胶原蛋白，位于关节面上或软骨细胞的胞浆中。可见本发明处理液允许一抗及二抗进入并特异性结合，同步实现了小鼠股骨头关节面及关节软骨整体器官的透明化处理及间接免疫荧光。FIG. 6 is a two-photon microscope image of the femoral head of the adult mouse in experiment 6 after the treatment solution kit of the present invention is transparentized and the whole tissue is indirectly immunolabeled with II-collagen in articular cartilage. The arrows in the figure show the type II collagen labeled with anti-II-collagen antibody, which is located on the articular surface or in the cytoplasm of chondrocytes. It can be seen that the treatment liquid of the present invention allows the primary and secondary antibodies to enter and specifically bind, and simultaneously realizes the transparent treatment and indirect immunofluorescence of the articular surface of the femoral head and the entire cartilage of the mouse.
本发明的技术方案至少具有以下优点之一：The technical solution of the present invention has at least one of the following advantages:
1、本发明所用处理液，37℃水浴恒温浸泡样本，没有繁杂的预处理和处理过程，操作简单；1. The treatment solution used in the present invention is a 37 ° C water bath soaking sample at a constant temperature, without complicated pretreatment and processing procedures, and simple operation;
2、本发明可使不同动物(大鼠及小鼠等)、不同器官(脑，脊髓，关节软骨，荷瘤肝等)器官透明并免疫标记；2. The present invention can make organs of different animals (rats, mice, etc.) and different organs (brain, spinal cord, articular cartilage, tumor-bearing liver, etc.) transparent and immunologically labeled;
3、本发明所用处理液可与荧光标记的一抗，一抗及荧光标记的二抗配制成整体免疫荧光溶液，可在整体器官水平实现同时透明化及免疫荧光标记的目的(见图1-6)。3. The treatment solution used in the present invention can be formulated with a fluorescently labeled primary antibody, a primary antibody, and a fluorescently labeled secondary antibody to form an overall immunofluorescent solution, which can achieve the purpose of simultaneous transparency and immunofluorescent labeling at the level of the entire organ (see Figure 1- 6).
以上仅为本发明的优选实施例而已，并非限定发明保护范围，凡在本发明的精神和原则之内的任何修改、替换或改进等均包含在本发明的保护范围中。The above are only preferred embodiments of the present invention, and do not limit the scope of protection of the invention. Any modification, replacement, or improvement within the spirit and principle of the present invention is included in the scope of protection of the present invention.

Claims (9)

  1. 一种处理液组合物，包含：A treatment liquid composition comprising:
    透明化处理液；和Clearing treatment fluid; and
    荧光标记抗体，所述荧光标记抗体与所述透明化处理液的体积比为1:10至1:500，其中，所述透明化处理液包含：A fluorescently labeled antibody, wherein the volume ratio of the fluorescently labeled antibody to the clearing treatment solution is 1:10 to 1: 500, wherein the clearing treatment solution includes:
    糖类；carbohydrate;
    尿素；Urea
    非离子表及面活性剂，所述非离子表面活性剂包括体积比为5:3至1:5的曲拉通和吐温混合物。Non-ionic surface and surface active agent, the non-ionic surface active agent comprises a triton and a Tween mixture with a volume ratio of 5: 3 to 1: 5.
  2. 根据权利要求1所述的处理液组合物，其中所述糖类为果糖或蔗糖中的至少一种；和/或，所述曲拉通包括曲拉通X-100和曲拉通X-114；和/或，所述吐温包括吐温20，吐温40和吐温60。The treatment liquid composition according to claim 1, wherein the saccharide is at least one of fructose or sucrose; and / or, the trolaton comprises trolaton X-100 and tralaton X-114 And / or, the Tween includes Tween 20, Tween 40 and Tween 60.
  3. 根据权利要求1所述的处理液组合物，其中每100ml所述透明化处理液包含：20-30g的尿素，10-15ml的非离子表面活性剂，40-60g的糖类，0-20ml的氨类，0-2ml的抗氧化剂，余量为水；优选地，随着糖类含量的增加，尿素含量也增加，或者随着糖类含量的减少，尿素含量也减少。The treatment liquid composition according to claim 1, wherein each 100 ml of the clearing treatment liquid comprises: 20-30 g of urea, 10-15 ml of a nonionic surfactant, 40-60 g of sugars, and 0-20 ml of Ammonia, 0-2ml antioxidant, the balance is water; preferably, as the sugar content increases, the urea content also increases, or as the sugar content decreases, the urea content also decreases.
  4. 根据权利要求1所述的处理液组合物，其中抗体包括以下抗体中的至少一种：The treatment liquid composition according to claim 1, wherein the antibody comprises at least one of the following antibodies:
    抗甲胎蛋白抗体(anti-AFP)，分子量约65kD，优选使用浓度范围为5-25μg/ml；Anti-fetoprotein antibody (anti-AFP), with a molecular weight of about 65kD, preferably in a concentration range of 5-25 μg / ml;
    抗生存素抗体(anti-Survivin)，分子量约16kD，优选使用浓度范围为5-25μg/ml；和Anti-Survivin antibody, with a molecular weight of about 16 kD, preferably in a concentration range of 5-25 μg / ml; and
    抗绿色荧光蛋白抗体(anti-GFP)，分子量约27kD，优选使用浓度范围为10-100ug/ml。Anti-green fluorescent protein antibody (anti-GFP), with a molecular weight of about 27kD, preferably in a concentration range of 10-100 ug / ml.
  5. 一种处理液试剂盒，包含：A processing solution kit includes:
    第一部分，包括：透明化处理液和按1:10至1:500体积比与透明化处理液混合的一抗；和The first part includes: a transparent treatment liquid and a primary antibody mixed with the transparent treatment liquid in a volume ratio of 1:10 to 1: 500; and
    第二部分，包括：透明化处理液和按1:10至1:500体积比与透明化处理液混合的带荧光标记的二抗，The second part includes: a transparent solution and a fluorescently labeled secondary antibody mixed with the transparent solution in a volume ratio of 1:10 to 1: 500,
    其中所述透明化处理液为根据权利要求1-3中任一项所述的透明化处理液。The clearing treatment liquid is the clearing treatment liquid according to any one of claims 1-3.
  6. 根据权利要求5所述的处理液试剂盒，其中所述的一抗包括：The processing solution kit according to claim 5, wherein the primary antibody comprises:
    抗哺乳动物雷帕霉素靶蛋白(anti-mTOR)抗体，兔源，分子量约289kD，优选使用浓度范围为10-50μg/ml；Anti-mammalian rapamycin target protein (anti-mTOR) antibody, rabbit origin, molecular weight is about 289kD, preferably in a concentration range of 10-50 μg / ml;
    抗β-Ⅲ型微管蛋白抗体(anti-β-Ⅲ Tubulin)，小鼠来源，分子量约55kD，优选使用浓度范围为10-50μg/ml；Anti-β-Ⅲ tubulin antibody (anti-β-Ⅲ Tubulin), derived from mice, with a molecular weight of about 55kD, preferably in a concentration range of 10-50 μg / ml;
    抗神经元特异性核蛋白抗体(anti-NeuN)，兔源，分子量约34kD，优选使用浓度范围为10-200μg/ml；或Anti-neuron-specific nucleoprotein antibody (anti-NeuN), rabbit origin, with a molecular weight of about 34 kD, preferably in a concentration range of 10-200 μg / ml; or
    抗Ⅱ型胶原蛋白抗体(anti-Ⅱ collagen)，分子量约142kD，小鼠来源，优选使用浓度范围为10-200μg/ml，Anti-type II collagen antibody (anti-II collagen), molecular weight about 142kD, mouse origin, preferably used in the concentration range of 10-200μg / ml,
    所述的带荧光标记的二抗是能够与一抗特异性结合(抗小鼠或抗兔)的免疫球蛋白(IgG)，其携带荧光标记。The fluorescently labeled secondary antibody is an immunoglobulin (IgG) capable of specifically binding to the primary antibody (anti-mouse or anti-rabbit), which carries a fluorescent label.
  7. 一种生物器官透明化同时进行免疫标记的方法，包括用权利要求1-4中任一项所述的处理液组合物浸泡生物组织器官；优选地，浸泡在37℃恒温进行1-7天。A method for transparentizing biological organs and performing immunolabeling at the same time, comprising soaking biological tissue organs with the treatment solution composition according to any one of claims 1 to 4;
  8. 一种生物器官透明化同时进行免疫标记的方法，包括用根据权利要求5-6中任一项所述的处理液试剂盒处理生物组织器官，其中A method for transparentizing biological organs and performing immunolabeling at the same time, comprising treating biological tissue organs with the treatment solution kit according to any one of claims 5 to 6, wherein
    先用第一部分浸泡生物组织器官，优选地，浸泡在37℃恒温进行1-4天；First immerse the biological tissues and organs with the first part, preferably, soak at 37 ° C for 1-4 days;
    然后，用第二部分浸泡生物组织器官，优选地，浸泡在37℃恒温进行1-3天。Then, the second part is used to soak the biological tissues and organs, preferably at 37 ° C for 1-3 days.
  9. 根据权利要求7或8所述的生物器官透明化同时进行免疫标记的方法，其中所述生物组织器官包括脑、脊髓、关节软骨以及脏器。The method according to claim 7 or 8, wherein the biological tissue organ is transparent, and immunolabeling is performed, wherein the biological tissue organ includes brain, spinal cord, articular cartilage, and organs.
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