阅读说明：本技术 一种全量组织免疫荧光方法和应用 (Full-tissue immunofluorescence method and application ) 是由 许佳祺 李全林 周平红 于 2021-09-16 设计创作，主要内容包括：本发明公开了一种全量组织免疫荧光方法和应用,属于组织免疫荧光技术领域。该免疫荧光方法包括获取待荧光染色的全量组织,甲醛固定液固定过夜后进行免疫荧光染色,其中全量组织来源于经心脏灌注后的实验小鼠。本发明通过对小鼠心脏灌注后获取的全量组织进行免疫荧光染色,有效冲洗血管中残留的血细胞,减少非特异性染色和血管内免疫细胞对染色结果的干扰,可实现细胞立体化观察,避免计数不准确和观察不确切的缺陷,克服传统免疫荧光染色无法对分布稀疏和胞体较大的细胞有效观察和计数的问题。(The invention discloses a full-tissue immunofluorescence method and application, and belongs to the technical field of tissue immunofluorescence. The immunofluorescence method comprises the steps of obtaining the total tissue to be subjected to fluorescent staining, fixing the tissue overnight by formaldehyde fixing solution, and then carrying out immunofluorescence staining, wherein the total tissue is derived from an experimental mouse subjected to heart perfusion. According to the invention, the immunofluorescence staining is carried out on the whole amount of tissues obtained after the heart of the mouse is perfused, so that the residual blood cells in the blood vessel are effectively washed, the interference of nonspecific staining and immune cells in the blood vessel on staining results is reduced, the three-dimensional observation of the cells can be realized, the defects of inaccurate counting and inaccurate observation are avoided, and the problem that the traditional immunofluorescence staining cannot effectively observe and count the cells with sparse distribution and large cell bodies is solved.)

1. A full-scale tissue immunofluorescence kit is characterized by comprising a confining liquid, a cleaning liquid, a primary anti-diluent, a secondary anti-diluent, a DAPI fluorescent dye and an anti-quenching confining tablet; wherein:

the blocking solution contained 10% BSA and 0.5% triton;

the cleaning solution is PBS solution containing 0.5% triton;

the primary antibody dilution was 5% BSA and 0.5% triton in PBS and the secondary antibody dilution was 2.5% BSA and 0.5% triton in PBS.

2. The use of the full-volume tissue immunofluorescence kit of claim 1 in the stereoscopic observation of gut neurons or immune cells.

3. The use of claim 2, wherein the gut-gut neurons are esophageal-gut myo-layer neurons and the immune cells are macrophages.

4. A full-tissue immunofluorescence method is characterized by comprising the following steps:

(1) obtaining a total amount of tissues to be subjected to fluorescent staining, and fixing the tissues overnight by using 4% formaldehyde fixing solution;

(2) the whole fixed tissue is subjected to fluorescent staining by the whole tissue immunofluorescence kit according to claim 1, and the steps are as follows:

(a) washing with 0.5% triton-containing PBS for 30min for 3 times;

(b) blocking with blocking solution containing 10% BSA and 0.5% triton at room temperature for 2 hours;

(c) washing with 0.5% triton-containing PBS for 30min for 3 times;

(d) primary antibody was diluted with 5% BSA and 0.5% triton in PBS and incubated overnight at 4 degree freezer;

(e) washing with 0.5% triton-containing PBS for 30min for 3 times;

(f) the secondary antibody was diluted with 2.5% BSA and 0.5% triton in PBS and incubated at room temperature in the dark for 2 hours;

(g) washing with 0.5% triton-containing PBS for 30min for 3 times;

(h) incubating with DAPI, namely dye solution, at room temperature in dark for 20 min;

(i) washing with 0.5% triton-containing PBS for 30min for 3 times;

(j) taking out the whole treated tissue, spreading the tissue on a glass slide, drying the tissue in the air, dripping an anti-quenching sealing agent for sealing, and storing the tissue in a dark place;

wherein the total tissue is derived from experimental mice after cardiac perfusion.

5. The method of claim 3, wherein the total tissue is gut neurons or immune cells.

6. The method of claim 5, wherein the gut neurons are esophageal gut layer neurons and the immune cells are macrophages.

7. The method for full-tissue immunofluorescence according to any one of claims 4 to 6, wherein the method for obtaining full-tissue comprises the steps of fixing, dissecting and left ventricle perfusion of the mouse, and specifically comprises the following steps: the abdominal cavity of the mouse is injected with Avermectin with the dose of 20mg/kg for anesthesia, the four limbs of the mouse are fixed quickly after the mice take effect, the abdominal cavity is opened layer by layer, the diaphragm is opened after the sternum angle to enter the thoracic cavity, the thoracic cavity is opened to the two sides, the sternum angle is clamped by vascular forceps after the complete separation to be turned upwards, and the heart is exposed; inserting a scalp needle into the apex of the heart, fixing the scalp needle by the right hand to prevent slippage, cutting the right auricle by the left hand with an ophthalmological scissors, connecting the scalp needle with a 20ml syringe, slowly injecting 20ml of normal saline, continuously injecting 10ml of 4% paraformaldehyde after the color of the liver gradually changes from red to white, and ending the process.

8. Use of the full-scale tissue immunofluorescence method of any one of claims 4 to 6 for the visualization of cell stereopsis.

9. The use of claim 8, wherein the cell is a gut neuron or an immune cell.

10. Use according to claim 8 or 9, wherein said cell stereoscopic observation comprises observing the spatial distribution and counting of said cells.

Technical Field

The invention belongs to the technical field of tissue immunofluorescence, and particularly relates to a full-tissue immunofluorescence method and application, which are particularly used for three-dimensionally observing the spatial distribution and counting of different cell populations in a tissue.

Background

The conventional mouse tissue immunofluorescence staining is based on tissue of a thin section (2-3 mu m), and the tissue quantity corresponding to the section is limited for the tissue with a small cross section (such as esophagus), so that the traditional method is difficult to effectively obtain a satisfactory staining result for cells with sparse distribution or large cell bodies, the section plane is usually difficult to capture the target cells, and the cells in the section plane cannot be effectively counted. In addition, for immune cells, non-specific staining of erythrocytes in blood vessels interferes with fluorescence, and the immune cells stained in blood vessels need to be eliminated, and the conventional method generally includes obtaining tissues after killing mice, and blood stagnation in blood vessels, which can not avoid the defects.

Disclosure of Invention

The invention mainly aims to provide a full-tissue immunofluorescence method, which can be used for effectively observing and counting cells with sparse distribution and large cell bodies in tissues by reducing the influence of blood cells in animal blood vessels on staining and obtaining the full-tissue after heart perfusion without slicing treatment.

The invention also aims to provide the application of the tissue immunofluorescence method in three-dimensional observation of cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a full-tissue immunofluorescence kit, which comprises a confining liquid, a cleaning liquid, a primary anti-diluent, a secondary anti-diluent, a DAPI (4', 6-diamidino-2-phenylindole) fluorescent dye and an anti-quenching confining tablet; wherein:

the blocking solution contained 10% BSA and 0.5% triton;

the cleaning solution is PBS solution containing 0.5% triton;

the primary antibody dilution was 5% BSA and 0.5% triton in PBS and the secondary antibody dilution was 2.5% BSA and 0.5% triton in PBS.

The invention provides an application of the full-tissue immunofluorescence kit in three-dimensional observation of gastrointestinal neurons or immunocytes.

Preferably, the gut enteric neuron is an esophageal enteric layer neuron, or the immune cell is a macrophage.

The invention also provides a full-tissue immunofluorescence method, which comprises the following steps:

(1) obtaining a total amount of tissues to be subjected to fluorescent staining, and fixing the tissues overnight by using 4% formaldehyde fixing solution;

(2) fluorescence staining of the total tissue: the fixed total tissue is subjected to fluorescent staining by the total tissue immunofluorescence kit, and the steps are as follows:

(a) washing with 0.5% triton-containing PBS for 30min for 3 times;

(b) blocking with blocking solution containing 10% BSA and 0.5% triton at room temperature for 2 hours;

(c) washing with 0.5% triton-containing PBS for 30min for 3 times;

(d) primary antibody was diluted with 5% BSA and 0.5% triton in PBS and incubated overnight at 4 degree freezer;

(e) washing with 0.5% triton-containing PBS for 30min for 3 times;

(f) the secondary antibody was diluted with 2.5% BSA and 0.5% triton in PBS and incubated at room temperature in the dark for 2 hours;

(g) washing with 0.5% triton-containing PBS for 30min for 3 times;

(h) incubating with DAPI, namely dye solution, at room temperature in dark for 20 min;

(i) washing with 0.5% triton-containing PBS for 30min for 3 times;

(j) taking out the whole treated tissue, spreading the tissue on a glass slide, drying the tissue in the air, dripping an anti-quenching sealing agent for sealing, and storing the tissue in a dark place;

wherein the total tissue is derived from experimental mice after cardiac perfusion.

Preferably, the full amount of tissue is gut neurons or immune cells.

Preferably, the method for obtaining the total amount of tissue comprises the steps of fixing, dissecting and perfusing the left ventricle of the mouse, and specifically comprises the following steps: the abdominal cavity of the mouse is injected with Avermectin with the dose of 20mg/kg for anesthesia, the four limbs of the mouse are fixed quickly after the mice take effect, the abdominal cavity is opened layer by layer, the diaphragm is opened after the sternum angle to enter the thoracic cavity, the thoracic cavity is opened to the two sides, the sternum angle is clamped by vascular forceps after the complete separation to be turned upwards, and the heart is exposed; inserting a scalp needle into the apex of the heart, fixing the scalp needle by the right hand to prevent slippage, cutting the right auricle by the left hand with an ophthalmological scissors, connecting the scalp needle with a 20ml syringe, slowly injecting 20ml of normal saline, continuously injecting 10ml of 4% paraformaldehyde after the color of the liver gradually changes from red to white, and ending the process.

The invention also provides the application of the full-tissue immunofluorescence method in cell three-dimensional observation.

Preferably, the cell is a gut enteric neuron or an immune cell.

More preferably, the gut enteric neuron is an esophageal enteric layer neuron and the immune cell is a macrophage.

Preferably, the cell stereotactic observation comprises observing the spatial distribution and count of different cell populations.

Compared with the prior art, the invention has the beneficial effects that:

(1) the full tissue immunofluorescence method of the invention carries out immunofluorescence staining on the full tissue after heart perfusion, utilizes the blood supply passage of the heart to tissue organs, and the physiological saline and the stationary liquid injected from the left ventricle can reach the tissue capillary, effectively washes the residual blood cells in the blood vessel, and reduces the interference of nonspecific staining and the immune cells in the blood vessel to the staining result.

(2) The invention utilizes the penetration of triton to carry out full-scale dyeing on the whole tissue, obtains target cells to the maximum extent, realizes the three-dimensional observation of the cells by confocal multilayer scanning, avoids the defects of inaccurate counting and inaccurate observation, and overcomes the problem that the traditional immunofluorescence dyeing can not effectively observe and count the cells with sparse distribution and larger cell bodies.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is (a) conventional immunofluorescent staining of tissue sections: the rare neurons can be counted and can not be compared; (b) the invention comprises the following steps: neurons can be counted after stacking of multiple layers.

FIG. 2 is (a) conventional immunofluorescent staining of tissue sections: the macrophage has incomplete form and strong nonspecific staining; (b) the invention comprises the following steps: macrophage foot-bulge-like morphology.

FIG. 3 is a picture of the procedure for obtaining full-volume tissue by heart perfusion in experimental mice, wherein: (1) fixing; (2) dissecting; (3) indicating the left atrial appendage; (4) left ventricular perfusion; (5) the liver whitens after successful perfusion; (6) raising tail when pouring the fixing liquid.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention are clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.

The esophageal enteromyocyte neurons and macrophages are exemplified in the following examples.

Example 1

In this example, the whole tissue is obtained after heart perfusion of experimental mice (as shown in fig. 3), and the steps are as follows:

1. perfusion

On the 7 th day after the mice are administrated with tamoxifen, the mice are anesthetized by Abametin with the intraperitoneal injection dose of 20mg/kg, the limbs of the mice are fixed quickly after the mice take effect, the abdominal cavity is opened layer by layer, the diaphragm is opened from the back of the sternum angle, the thoracic cavity is entered into the thoracic cavity, the thoracic cavity is opened towards the two sides, and after full separation, the sternum angle is clamped by vascular clamps to be turned upwards to expose the heart. Inserting scalp needle into apex of heart, fixing scalp needle with right hand to prevent slippage, and cutting right auricle with left-hand ophthalmology scissors. The scalp needle is connected with a 20ml syringe, and 20ml of normal saline is injected slowly, so that the color of the liver gradually changes from red to white, and the effect of perfusion is indicated. After the normal saline is filled, 10ml of 4% paraformaldehyde is continuously filled, and the tail of the mouse is stiff and tilted at the moment, which indicates that the fixation is effective.

2. Taking materials

After the perfusion fixation is finished, the esophagus is found along the stomach, the esophagus is fully separated, and the esophagus and the stomach are cut off at the middle section and the stomach bottom of the esophagus respectively. Cleaning esophagus and stomach contents by using normal saline, longitudinally cutting a lumen, fixing the head end and the tail end by using pins, separating a muscular layer and a mucous layer from one end, wherein the muscular layer is soft and the mucous layer is relatively tough, tearing the muscular layer from the mucous layer along a longitudinal axis after finding out a tissue gap, and quickly putting the obtained muscular tissue into 4% formaldehyde fixing solution for fixing.

Example 2

Immunofluorescent staining was performed on the total amount of tissue obtained in example 1, as follows:

1) fixing with 4% formaldehyde fixing solution overnight, washing with 0.5% triton-containing PBS solution for 3 times, each for 30 min;

2) blocking with blocking solution containing 10% BSA and 0.5% triton at room temperature for 2 hours;

3) washing with 0.5% triton-containing PBS for 30min for 3 times;

4) diluting anti-mouse HuC/D and anti-mouse F4/80 with 5% BSA and 0.5% triton in PBS at a dilution ratio of 1: 400, 4 degree refrigerator incubation primary antibody overnight.

5) Washing with 0.5% triton-containing PBS for 30min for 3 times;

6) the anti-goat secondary-488 and anti-rat-Cy 3 were diluted with PBS solution containing 2.5% BSA and 0.5% triton at a dilution ratio of 1: 300. incubate for 2 hours at room temperature in the dark.

7) Washing with 0.5% triton-containing PBS for 30min for 3 times;

8) incubating with DAPI, namely dye solution, at room temperature in dark for 20 min;

9) washing with 0.5% triton-containing PBS for 30min for 3 times;

taking out the tissue, spreading the tissue on a glass slide, enabling the muscle to be parallel to the plane of the glass slide, drying the water in the air, dripping the anti-quenching sealing tablet, and storing the sealing tablet in the dark.

The manufactured full-fluorescence dyeing tissue slices are subjected to confocal multilayer scanning and fusion, and the method comprises the following steps: the secondary antibodies used for staining were switched on with corresponding lasers including 488, DAPI and Cy 3. First, the tissue piece is observed through an ocular lens, the dyeing condition is checked, and the position and the layer surface of the neuron are coarsely focused under the 488 laser condition. When observed in a 10-fold mirror preview mode, the slice is finely adjusted to a slice with high density distribution of both neurons and macrophages, and the slice is taken as the center, 10um upward is set as the upper bound, 10um downward is set as the lower bound, scanning is performed once every 1um, images are fused after scanning, and the number of neurons and the number of macrophages are counted, and the result is shown in fig. 1 and fig. 2.

In conclusion, the invention has the advantages over the traditional section staining when the histological research on the cells with sparse distribution and large cell bodies is required for the tissues with small cross sections of animals, and has use value for the research on digestive tract enteric neurons, immune cells and the like.