CN113640520A

CN113640520A - Application of tissue transparency method and histology method in combination for detecting bacteria in tumor

Info

Publication number: CN113640520A
Application number: CN202110810278.5A
Authority: CN
Inventors: 孙海涛; 李婷; 赵佳佳; 贺电; 骆韵豪
Original assignee: Southern Medical University Zhujiang Hospital
Current assignee: Southern Medical University Zhujiang Hospital
Priority date: 2021-07-16
Filing date: 2021-07-16
Publication date: 2021-11-12
Also published as: JP2024530072A; WO2023284820A1

Abstract

The invention relates to the technical field of detection, and particularly discloses an application of a tissue transparency method and a histology method in combination for detecting bacteria in tumors. The invention combines the tissue transparency method and the traditional histology method to break the limit (several microns) to the thickness of the sample, thereby avoiding the potential pollution on the surface of the thin slice; and a high-resolution and integral three-dimensional image of a large-size tissue sample can be completely constructed, so that three-dimensional visualization of bacteria in the tumor is realized. The invention selects the tissue with the thickness of 500 mu m, achieves optical transparency by using a tissue transparency method, and simultaneously marks the bacteria in the tumor tissue by combining a traditional histology method (such as an immunofluorescence marking method), thereby obtaining more accurate experimental results and laying a solid foundation for researching the direct interaction between microbial populations and tumor cells in a tumor microenvironment.

Description

Application of tissue transparency method and histology method in combination for detecting bacteria in tumor

Technical Field

The invention relates to the technical field of detection, in particular to an application of a tissue transparency method and a histology method in combination for detecting bacteria in tumors.

Background

In recent years, there have been increasing studies showing that the relationship between microorganisms and tumors is far greater than expected. With the progress of the tools for characterizing the microbiome, the influence of the human microecological system on the occurrence and development of tumors, immune response, therapeutic effect and prognosis gradually draws a great deal of attention. Infection-related cancers and the presence of intratumoral microorganisms have also been identified in a variety of tumor types, the latter being thought to promote tumor development by mechanisms such as direct induction of carcinogenesis, modulation of oncogenic molecular pathways, or modulation of the host immune system.

In recent years, there have been strong discussions about the presence of bacteria in tumors, whether bacteria favor a certain intratumoral microenvironment, and whether the abundance of bacteria is independent of their effects. "The human tumor composition of tumor type-specific intracellular bacteria" reported that Nejman et al performed comprehensive bacterial detection of seven solid tumors such as breast cancer, lung cancer, ovarian cancer, pancreatic cancer, melanoma, bone cancer and brain glioma by various methods. They found that bacterial LPS and 16s rRNA were detectable in all tumor types and that there was a significant intratumoral difference in bacterial abundance and type. However, this study is mainly based on immunohistochemical staining and fluorescence in situ hybridization and sequencing methods performed on paraffin sections, which are thin sections, to detect bacteria, and this method system is difficult to avoid the contamination problem that may be introduced during sample processing, and the destructive processing of these methods may cause misreading of the number and location of bacteria. Therefore, there is an urgent need for a more sophisticated methodology to provide more definitive evidence of the presence of microorganisms in tumors and to characterize the distribution and function of tumor microbiome.

At present, the direct interaction between microbial populations and tumor cells in a tumor microenvironment can be better understood by providing more powerful and intuitive evidence for microbial detection and three-dimensional reconstruction of thick tumor tissue samples. The tissue transparency technology can break the limit (several microns) of the tissue section technology on the thickness of a sample, avoid potential pollution on the surface of a thin section and facilitate the three-dimensional visualization of microbial populations in the whole tissue with single cell resolution. Therefore, the application of tissue transparency technology to the detection of microorganisms is a completely new attempt and is expected to provide a high-throughput, contamination-free, unbiased method for the detection of tumor microorganisms.

The tissue transparency technology is a technology which applies a water-soluble organic solvent or a hydrophilic reagent, performs permeabilization treatment on a fixed tissue in a soaking, electrophoresis or perfusion mode and the like, then applies a high-refractive-index medium to match the refractive index of the tissue, reduces light scattering, enables the tissue to be optically transparent, and further increases the imaging depth and the image contrast. On the premise of keeping the integrity of the tissue structure, the tissue transparency technology can realize three-dimensional imaging at the cell level, and avoids the loss of spatial structure information, so the tissue transparency technology is a key technology for opening the potential of an optical microscope and is one of the best selection schemes for drawing a whole organ or whole body cell map. Therefore, the tissue transparency technology is combined with the microscopic imaging technology, so that the process of volume imaging (whole body or organ imaging) is greatly promoted, the comprehensive understanding of a complete biological system is enhanced, and a strong and beneficial tool is provided for the field of biomedical research.

In recent years, tissue transparency technology has been rapidly developed, and various new transparency technologies including DISCO, Murray, uDISCO, Scale, SeeDB, category, CUBIC, iDISCO, FRUIT, TDE, pegsos, OPTIClear, etc. are continuously emerging, and different transparency technologies have specific tissue applicability. The transparent technology OPTIClear specially developed for human tissues can effectively transparent fresh and formaldehyde-fixed paraffin-embedded human brain tissues (5mm thick), well preserve ultrastructures, is compatible with various fluorescent dyes and lipophilic dyes, and can carry out high-resolution three-dimensional imaging on neuron projection and dendritic spine structures of the human brain tissues by combining with lipophilic dye tracing.

The Accu-OPTIClear technology is to make the tissue optically transparent, so as to increase the imaging depth and image contrast, however, the labeling of bacteria is still dependent on traditional histological methods such as Immunofluorescence (IF), and in situ Fluorescence hybridization (FISH), but the traditional histological methods and the currently emerging sequencing technologies are difficult to avoid the contamination problem that may be introduced during the sample processing, and the destructive processing of the methods may cause misreading of the number and location of bacteria. In addition, no report has been made in the prior art on the application of a tissue clearing method and a histological method in combination to detect bacteria in tumors or other related studies.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of the combination of the tissue transparency method and the histology method for detecting the bacteria in the tumor, the combination of the tissue transparency method and the traditional histology method can break the limit (several microns) on the thickness of a sample, thereby avoiding the potential pollution on the surface of a thin section; and a high-resolution and integral three-dimensional image of a large-size tissue sample can be completely constructed, so that three-dimensional visualization of bacteria in the tumor is realized, the overall appearance of the bacteria in a tumor microenvironment is shown, and a foundation is laid for researching the interaction relationship between tumor cells and microorganisms of a host in the future.

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

in a first aspect, the present invention provides the use of a tissue transparency method in combination with a histological method for the detection of bacteria in tumors.

Compared with the traditional histology method, the tissue transparency method and the traditional histology method are combined to obtain a more reliable result on detecting the bacteria in the tumor, the tissue prepared by the combined method can be three-dimensionally reconstructed by a confocal microscope, and the spatial distribution condition of the bacteria in the tumor tissue can be visually shown from a three-dimensional angle.

As a preferred embodiment of the use according to the invention, the tissue transparentization method comprises a hydrophobic transparentization method or a hydrophilic transparentization method. More preferably, the hydrophobic transparentization method includes, but is not limited to, 3DISCO, idsco, uDISCO, vDISCO, etc.; more preferably, the hydrophilic transparentization method includes, but is not limited to, Scale, SeeDB, CUBIC-X, Accu-OPTIClear, and the like.

On the basis of OPTIClear, the invention uses the Accu-OPTIClear transparentizing method to avoid excessive defatting, reduce the time for transparentizing tissues, reduce tissue damage and better preserve antigens.

As a preferred embodiment of the application of the invention, the histological method comprises one of an immunofluorescent labeling method, an immunoenzyme labeling method and a fluorescence in situ hybridization labeling method.

As a preferred embodiment of the application of the invention, the thickness of the tissue slice adopted in the process of detecting the bacteria in the tumor is 400-600 μm.

Most of the current histological detection methods of bacteria are immunohistochemistry and fluorescence in situ hybridization technologies of paraffin sections, but the paraffin sections are mostly thin sections (4-6 mu m), so the problem of surface pollution in the treatment process is difficult to avoid. The invention selects thick tissues (400-600 mu m), achieves optical transparency by using a tissue transparency method, and simultaneously marks bacteria in the tumor tissues by combining a traditional histology method (such as an immunofluorescence marking method), thereby obtaining more accurate experimental results and laying a solid foundation for researching the direct interaction between microbial populations and tumor cells in a tumor microenvironment.

More preferably, the tissue slices have a thickness of 500 μm.

When the thickness of the tissue slice is 500 mu m, the tissue transparency technology is utilized to achieve optical transparency, and simultaneously, the immunofluorescence method is combined to mark bacteria in the tumor tissue, so that the most accurate experimental result is obtained.

As a preferred embodiment of the use of the present invention, the tumor comprises at least one of human brain glioma, breast cancer, pancreatic cancer, melanoma.

In a second aspect, the present invention provides a method for detecting bacteria in tumor by combining a tissue transparency method and an immunofluorescent labeling method, which comprises the following steps:

1) obtaining a tissue slice with the thickness of 400-600 mu m, preparing an OPTIClear tissue transparent reagent, dissolving a detergent in the OPTIClear tissue transparent reagent to obtain a mixed solution, soaking the tissue slice in the mixed solution, wherein the volume ratio of the tissue slice to the mixed solution is more than 1:3, adding an autofluorescence quencher, washing the soaked tissue slice with a PBS solution, and adding a sealant for overnight incubation;

2) placing the tissue slices treated in the step 1) in a pore plate, adding a primary antibody diluent for incubation, washing the incubated tissue slices with a buffer solution, and adding a secondary antibody diluent for incubation;

3) adding 4', 6-diamidino-2-phenylindole to mark cell nucleus while adding a secondary antibody diluent for incubation, and then washing the tissue section by using the same buffer in the step 2);

4) washing the tissue slice treated in the step 3) with a PBS solution, adding an OPTIClear tissue transparent reagent, incubating in a dark place, and finally observing under a mirror and reconstructing a three-dimensional image.

More preferably, the detergent in step 1) is Sodium Dodecyl Sulfate (SDS) to increase the transparency and degree of discoloration of the tissue, and the washing solution may be of other kinds.

The tissue slices are soaked in the mixed solution (the volume ratio of the tissue slices to the mixed solution is more than 1:3), so that the optical transparency of the tissue slices can be realized; adding a sealant for sealing, so that the sealant is combined with sites with cross reaction in the tissue slices in advance, and the occurrence of false positive is reduced; the interference of autofluorescence in tissue can be eliminated by adding autofluorescence quenching agent into the system.

In the step 3), 4', 6-diamidino-2-phenylindole (DAPI) is added into the system to mark cell nucleus, so that the relative position of bacteria and cells can be observed. And 4) adding an OPTIClear tissue transparent reagent into the mixture to ensure that the tissue section is optically transparent and is easy to observe under a fluorescence microscope.

As a preferred embodiment of the detection method, in the step 2), the ratio of the primary antibody to the secondary antibody to the diluent is 1:100, the volume ratio of the tissue section to the diluent is 1:2-1:3, and the tissue section can be soaked in the diluent.

As a preferred embodiment of the detection method of the present invention, the primary antibody in step 2) is a lipopolysaccharide antibody of a monoclonal mouse, and the secondary antibody is an donkey anti-mouse IgG antibody coupled with Alexa Fluor Plus 594.

As a preferred embodiment of the detection method of the present invention, the primary antibody and the secondary antibody are added in step 2) for incubation for at least 2 days.

In the technical scheme of the invention, the primary antibody of lipopolysaccharide of the monoclonal mouse can be specifically combined with LPS of gram-negative bacteria, so as to identify and mark the bacteria; the donkey anti-mouse IgG antibody coupled to Alexa Fluor Plus 594 binds to the primary antibody, causing fluorescence on the bacteria labeled with the primary antibody.

In the early stage of the experiment, the inventor combines the tissue transparency method with the traditional histology method to still have some obstacles, mainly represented by antibody permeability during immunofluorescence labeling, and longer incubation time is needed because the antibody and the fluorophore have larger molecular weight and slow permeation speed in the tissue. Therefore, the inventor conducts experiments continuously, and finally selects a tissue section with the thickness of 400-600 μm for conducting experiments in order to achieve good immunostaining quality in a short incubation time. In addition, bacteria deep in the tissue are observed by performing tissue clearing treatment on thick tissue slices so as to eliminate potential pollution on the surface of the thin slices; in addition, the microbial detection by using the fluorescence labeling method needs to eliminate the interference of spontaneous fluorescent substances in tissues, wherein the spontaneous fluorescent substances are light naturally emitted by subcellular structures such as lipofuscin, mitochondria and lysosomes after the subcellular structures absorb light, and the forms and the sizes of the subcellular structures are difficult to be simply distinguished from bacteria. The inventor adopts a chemical reagent quenching method to eliminate the interference as much as possible, thereby increasing the accuracy of the experimental result.

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

1) compared with the traditional histology method, the method combining the tissue transparency method with the traditional histology method can break the limitation (several microns) on the thickness of the tissue sample, thereby avoiding the potential pollution on the surface of the thin slice and obtaining more credible results;

2) the tissue prepared by the method system combining the tissue transparency method and the traditional histology method can be three-dimensionally reconstructed by a confocal microscope or a multi-photon laser scanning microscope, and the spatial distribution condition of bacteria in tumor tissue can be visually shown from a three-dimensional angle;

3) in the combined method, a chemical reagent quenching method is adopted to eliminate the interference of spontaneous fluorescent substances in tissues as much as possible, so that the false positive result is reduced, and the accuracy of the experiment is improved.

Drawings

FIG. 1 is a comparison of three different types of tissue samples before and after they are rendered transparent by the detection method of the present invention;

FIG. 2 shows EGFP^Tg/+Laser scanning confocal microscopy of mouse brain tissue sections (FIG. 2A is EGFP^Tg/+A planar panoramic scan of a mouse brain tissue section; FIG. 2B is a diagram of neuronal processes and connections in EGFPTG/+ mice; FIG. 2C is a three-dimensional reconstructed EGFP^Tg/+Mouse neuron connectivity graph);

FIG. 3 is a multiphoton laser scanning microscope observation image of a human glioma specimen after tissue transparency and immunofluorescence labeling combined treatment;

FIG. 4 is a three-dimensional reconstructed image of a human glioma sample after multiphoton laser scanning combined with tissue transparency and immunofluorescence labeling;

FIG. 5 is a confocal microscope observation image of mice after intestinal tissue transparency and immunofluorescence labeling combined treatment;

FIG. 6 is a labeling diagram of immunohistochemical staining of paraffin sections of human brain glioma specimens, C57BL/6 mouse brain tissue and small intestine tissue specimens;

FIG. 7 is an immunofluorescent staining marker map of paraffin sections of human brain glioma samples, C57BL/6 mouse brain tissue and small intestine tissue samples;

FIG. 8 is an in situ fluorescence hybridization labeling chart of paraffin sections of human brain glioma samples, C57BL/6 mouse brain tissues and small intestine tissue samples.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

The experimental procedures used in the following examples are conventional ones unless otherwise specified, and the materials, reagents and the like used therein are commercially available.

Material pretreatment:

experimental groups: human brain glioma tissue (GBM)3 cases;

control group: intestinal tissues of positive control mice 1 case + brain tissues of negative control mice 2 cases.

The experimental animal source is as follows: 2C 57BL/6 mice (5-6 weeks old, 18-22g, male) and 1 EGFPTG/+ mouse (3 months old, male) were obtained from the southern medical university Zhujiang Hospital laboratory animal center (license number: SYXK (Guangdong) 2019-. All animal in vivo experiments in the invention are carried out by strictly following the welfare ethical principle of experimental animals.

Taking intestinal tissues and brain tissues of mice and pretreating the intestinal tissues and the brain tissues of the mice: EGFP^Tg/+Mice were intraperitoneally injected with sodium pentobarbital (120mg/kg) and euthanized by transvascular perfusion with 0.9% normal saline followed by perfusion fixation with 4% (w/v) Paraformaldehyde (PFA). Immediately dissected and brains harvested, fixed with 4% PFA at 4 ℃ for about 1 year. Immediately after sacrifice of 1C 57BL/6 mouse by cervical dislocation, brains were dissected and harvested and fixed with 4% PFA at 4 ℃ for about 2-3 weeks. A second C57BL/6 mouse was anesthetized by intraperitoneal injection of sodium pentobarbital (120mg/kg) and heart perfused with 10ml/min cold PBS (pH7.4) followed by perfusion with 10ml/min 4% PFA. After total bowel dissection, the bowel contents were rinsed clean with 4% PFA and fixed without shaking at 4 ℃ for 2 days. Before the tissue was cleared, the tissue was gently washed overnight at 4 ℃ with 3X 30ml of PBS/0.01% (w/v) to remove paraformaldehyde residues.

Human brain glioma tissue sample sources: samples were obtained from the southern medical university Zhujiang Hospital clinical biosample bank. Randomly selected 3 human brain glioma surgically excised specimens were 2 males and 1 female, with an age between 2 and 47 years. All of the above samples were from glioma patients who did not receive drug therapy or radiotherapy and chemotherapy. The tumor is excised in surgery, the specimen is immediately rinsed with sterile physiological saline and the solid tumor part is selected and placed in a sterile sealed bottle and fixed and preserved in 10% neutral formalin solution. All the above operations require aseptic operations. The samples were confirmed by a clinician and graded according to WHO classification (WHO grade II 2: #1, #3, WHO grade IV 1: # 2). The project was reviewed and approved by the medical ethics committee of the southern medical university, Zhujiang Hospital, ethics No. 2018-SJWK-004, 2020-YBK-001-02, and with informed consent of the patients. And the clinical biological sample library of the south medical university Zhujiang hospital has passed the preservation approval of the China human genetic resource office (the approval numbers [2017]2042 and [2020] BC 0019).

Pretreatment of human brain glioma tissues: a plurality of sites far from the tumor edge were randomly selected from the human glioma specimen, and the sample was punched out with a biopsy punch (Integra Miltex, 4mm) and cut with a vibrating microtome (DOSAKA, DTK-2ER01N) or a common blade to a human glioma tissue section with a thickness of 500 μm.

Example 1

A tissue transparency method and an immunofluorescence labeling method are jointly applied to a detection method of bacteria in brain glioma, and the method comprises the following steps:

1) OPTIClear tissue clearing reagent (20% (w/v) N-methylglucamine (Sigma-Aldrich #66930), 32% (w/v) isohexanol (Sigma-Aldrich # D2158), and 25% -Thiodiethanol (TDE) (Sigma-Aldrich #88559) solutions were mixed, pH of the solutions was adjusted to 7 with concentrated hydrochloric acid, and Sodium Dodecyl Sulfate (SDS) detergent was dissolved in OPTIClear tissue clearing reagent to obtain SDS-OPTIClear mixture (SDS: OPTIClear ═ 4: 100) (ii) a

2) Transferring the obtained brain glioma tissue section into a sterile EP tube, adding SDS-OPTIClear mixed solution to soak the brain glioma tissue section in the SDS-OPTIClear mixed solution, incubating for 1-3 days at 37 ℃, then dropwise adding a proper amount of Sudan black solution at room temperature, shaking for 2 hours in a dark place, washing the soaked tissue section with PBS solution for 3 x10 minutes, incubating in BSA sealant at 37 ℃ overnight, removing the BSA sealant and washing the tissue with PBS solution;

3) placing the tissue slices treated in the step 2) into a 24-hole plate, adding lipopolysaccharide antibody (primary antibody) diluent of a monoclonal mouse for incubation, wherein the ratio of the primary antibody to the diluent is 1:100, volume ratio of tissue section to lipopolysaccharide antibody dilution of monoclonal mouse is 1:2, incubation at 37 ℃ for 2 days; the lipopolysaccharide antibody diluent after incubation of tissue sections with 0.2% PBS-Tween in the shaking table washing 6 x 30 minutes, leave the last washing liquid 37 degrees C overnight;

4) placing the tissue slices treated in the step 3) into a 48-well plate, adding donkey anti-mouse IgG (secondary antibody) coupled with Alexa Fluor Plus 594, wherein the ratio of the secondary antibody to the diluent is 1:100, the volume ratio of tissue sections to dilutions of donkey anti-mouse IgG antibody coupled to Alexa Fluor Plus 594 was 1:2, simultaneously adding 1 mu g/ml of 4', 6-diamidino-2-phenylindole to mark cell nuclei, incubating for 1 day at 37 ℃ in the dark, and washing tissues for 6X 30 minutes by using a 0.2% PBST solution;

5) gently blotting the solution around the tissue section treated in the step 4) with Kimwipes paper, placing the tissue section in a sterile EP tube, adding an OPTIClear solution with at least 3 times of volume to completely immerse the tissue section, and incubating the tissue section in the dark at 37 ℃ for about 15 hours;

6) and (4) observing under a mirror: taking a cell culture dish with the diameter of 60mm, flatly putting a stained tissue slice sample into the dish, adding a few milliliters of OPTIClear solution to enable the stained tissue slice sample to immerse the front end of an objective lens, placing the dish under a multiphoton laser scanning microscope (Olympus FVMPE-RS, Japan), selecting an excitation wavelength of 750nm (DAPI) and 980nm (Alexa Fluor Plus 594) to observe tissues, and using an objective lens of X10 objective lens (XLPN 10XSVMP,10X/0.6 NA);

7) image acquisition and three-dimensional reconstruction: for a glioma sample, observing and shooting an area far away from the edge of the sample under a 10-time objective lens, selecting Begin \ End under a Z-stack menu to set a start and stop point mode, and browsing the whole tissue layer to determine the start and stop points during image preview. Setting a light cutting interval of 1 μm, selecting an area of the middle layer of 100 μm, magnifying by two times from zoom, and performing scanning imaging layer by layer at a resolution of 1024x 1024; stacking the obtained images in iMaris software for three-dimensional reconstruction;

all operations are performed in a biosafety cabinet, and reagents and consumables are subjected to filtration sterilization and high-temperature sterilization. The tissue sample is transferred and washed gently to avoid damaging the tissue.

Wherein, when the sample is 3 cases of human brain glioma tissues, the detection method is adopted as an experimental group; when the samples are intestinal tracts of 1 positive control group mouse and brain tissues of 2 negative control group mice, the detection method is adopted as a control group.

The experimental results are as follows: referring to FIG. 1, EGFP with a thickness of 1-2mm^Tg/+The mouse brain tissue and the C57BL/6 mouse brain tissue section are treated by the method, the tissue transmittance is greatly improved by naked eyes, and the form change is small. The human glioma sections (with the thickness of 500 mu m, 1mm and 2mm) have smaller improvement on the transmittance after the same treatment than the former two sections, and the transmittance is better when the thickness is smaller, and 500 mu m is selected as the sampling and treatment thickness of the human glioma sample. Transparentized EGFP^Tg/+The brain tissue section of the mouse can observe the basic morphology and processes of the neurons and reconstruct the 3D structure thereof under a confocal microscope (refer to FIG. 2).

Three human glioma specimens (500 μm thick) were subjected to immunofluorescence staining with an anti-Lipopolysaccharide (LPS) antibody and DAPI counterstaining, then to tissue clearing and autofluorescence quenching, followed by observation under a multiphoton laser scanning microscope (10-fold mirror), with contamination carefully controlled throughout the treatment.

Referring to FIG. 3, specific red signals (indicated by arrows) of bacterial LPS were observed at different levels inside human glioma specimens with higher surface density and lower internal density, indicating that the positive signals have a diameter of about 0.7-2.0 μm, can be fusiform, circular, etc., and are distributed in the paranuclei and intercellular spaces.

Referring to fig. 4, in order to eliminate potential bacterial contamination on the surface of the sample, the tissue deep part is taken to be 100 μm for reconstruction and analysis, and the spatial distribution of the bacterial LPS specific signals in the human glioma sample can be visually seen, as observed in planar scanning, the LPS signals are mostly scattered in isolated points and are irregular in shape.

The effectiveness of LPS antibodies of selected bacteria was verified by observing under a microscope the presence of specific signals for LPS and LTA in the interstitial space of the intestinal villi of mice, with reference to FIG. 5(A) and FIG. 5(B) for small intestinal tissue before and after clearing and the arrows in FIG. 5(C) for marked bacteria in the intestinal sample of mice. However, the immunofluorescence morphology of the mouse intestinal bacteria is obviously different from that of human brain glioma, and is different from the typical characteristics and complete contour of gram-negative bacteria presented by the former, and the latter is often scattered and distributed, has irregular morphology and can be fusiform or round. This may be due to staining deviations caused by changes and disruptions in bacterial morphology during tissue fixation and permeabilization. Meanwhile, research indicates that in tumors, bacteria are often in a cell wall defect state or L-type bacteria, and the detected LPS can be phagocytosed bacterial fragments.

Example 2

An immunohistochemical method (IHC) applied to a detection method of bacteria in human brain glioma, comprising the following steps:

(1) preparation of paraffin sections: dehydrating the fixed human brain glioma sample, and embedding the sample in paraffin (dehydration: cherry blossom, VIPJ-JR; embedding: cherry blossom, TEC-5) to obtain a continuous section with the thickness of 4 mu m (Leica, RM 2245);

(2) paraffin section dewaxing to water: sequentially placing the slices in xylene I15min, xylene II 15min, xylene III 15min, anhydrous ethanol I5 min, anhydrous ethanol II 5min, 85% ethanol 5min and 75% ethanol 5min, and washing with distilled water;

(3) antigen retrieval: placing the tissue slices in a repairing box filled with citric acid antigen repairing buffer solution (pH6.0) in a microwave oven for antigen repairing, stopping heating for 8min until boiling, maintaining the temperature, and turning to low and medium heat for 7min to prevent excessive evaporation of the buffer solution, and cutting into dry slices; after natural cooling, the slide is placed in a PBS solution (PH7.4) and is shaken and washed on a decoloring shaker for 3 times, 5min each time;

(4) blocking endogenous peroxidase: placing the tissue slices into a hydrogen peroxide solution with the concentration of 3%, incubating at room temperature in a dark place for 25min, placing the slides into a PBS solution (PH7.4), and shaking and washing on a decolorizing shaker for 3 times, 5min each time;

(5) and (3) sealing: dripping 3% BSA solution (confining liquid) into the combined ring to uniformly cover the tissue, and sealing at room temperature for 30min to ensure that the confining liquid is combined with sites with cross reaction in the tissue in advance, thereby reducing the occurrence of false positive;

(6) primary antibody incubation: gently throw off the blocking solution, and drop 1: primary antibodies of 1000 monoclonal mouse lps (lipopolysaccharide) antibody (hycult biotech, WN 1222-5) and 1:1000 monoclonal mouse lta (lipoteichoic acid) antibody (GeneTex, GTX16470), sections were placed flat in a wet box and incubated overnight at 4 ℃ (small amount of water added in wet box to prevent evaporation of antibody);

(7) and (3) secondary antibody incubation: placing the tissue section after primary antibody incubation in PBS (PH7.4) and washing for 5min each time by shaking on a decolorizing shaker for 3 times; dripping an HRP-labeled donkey anti-mouse IgG antibody into the ring to cover the tissue after the section is slightly dried, and incubating for 50min at room temperature;

(8) DAB color development: placing the tissue section incubated by the secondary antibody in a PBS solution (PH7.4) and washing for 5min for 3 times by shaking on a decoloration shaking table; dripping a DAB color developing solution which is prepared freshly into the ring after the section is slightly dried, controlling the color developing time under a microscope, wherein the positive color is brown yellow, and flushing the section with tap water to stop color development;

(9) counterstaining cell nuclei: 3min or so by using the tissue slices of the hematoxylin counterstaining step (8), washing with tap water, differentiating the hematoxylin differentiation solution for several seconds, washing with tap water, returning the hematoxylin to blue by using the hematoxylin differentiation solution, and washing with running water;

(10) dewatering and sealing: placing the tissue slices in 75% alcohol for 5min, 85% alcohol for 5min, anhydrous ethanol I for 5min, anhydrous ethanol II for 5min, n-butanol for 5min, and xylene I for 5min, dehydrating, air drying, and sealing with neutral gum.

Example 3

An immunofluorescence method (IF) is applied to a detection method of bacteria in human brain glioma, and comprises the following steps:

(1) preparing a tissue section: dehydrating the fixed human brain glioma sample, and embedding the sample in paraffin (dehydration: cherry blossom, VIPJ-JR; embedding: cherry blossom, TEC-5) to obtain 4 μm-thick continuous sections (Leica, RM2245), namely paraffin sections;

(2) paraffin section dewaxing hydration: sequentially placing the slices in xylene I10min, xylene II 10min, xylene III 10min, anhydrous ethanol 5min, 95% ethanol 5min, 85% ethanol 5min and 75% ethanol 5min, and washing with distilled water for 3 times, each for 5 min;

(3) antigen retrieval: placing the tissue slices in a repairing box filled with citric acid antigen repairing buffer solution (pH6.0) in a microwave oven for antigen repairing, stopping heating for 5min until boiling, maintaining the temperature, and turning to middle and low heat for 5min to prevent excessive evaporation of the buffer solution and cutting into dry slices; after natural cooling, the slide is placed in a PBS solution (PH7.4) and is shaken and washed on a decoloring shaker for 3 times, 5min each time;

(4) autofluorescence quenching: dropwise adding tissue autofluorescence quencher A solution (Sericebio, G1221), incubating at room temperature for 30min, and washing with pure water for 5 min;

(5) and (3) sealing: the tissue sections were then blocked with 3% BSA (biofloxx, 4240GR025) for 2h, and then the serum was decanted;

(6) primary antibody incubation: the method comprises the following steps of 1:1000 monoclonal mouse LPS (lipopolysaccharide) antibody (HycultBiotech, WN 1222-5) is incubated at 4 ℃ for 12-18h, and rewarming at room temperature for 30 min;

(7) and (3) secondary antibody incubation: incubate donkey anti-mouse IgG antibody (Invitrogen, AB-2762826) coupled with 1:1000 Alexa Fluor Plus 594 at 37 ℃ at constant temperature;

(8) autofluorescence quenching: washing with PBS solution for 3 times, each for 3min, adding tissue autofluorescence quencher B solution (Sericebio, G1221) dropwise to the tissue slice, standing at room temperature for 5min, and washing with flowing water for 3 min;

(9) counterdyeing and mounting: finally, nuclei were counterstained with DAPI (ABCAM, ab104139), sealed with nail polish, and examined microscopically after 15 min.

Example 4

A Fluorescence In Situ Hybridization (FISH) method is applied to a detection method of bacteria in human brain glioma, and a direct fluorescence bacteria in situ hybridization detection kit (EUB338 probe) is used for detection, and the method comprises the following steps:

(1) paraffin section dewaxing hydration: baking paraffin slices at 72 deg.C for 2 hr, dewaxing with xylene, and sequentially hydrating with anhydrous ethanol, 85% ethanol, and 70% ethanol for 5 min; soaking in PBS solution for 2 times at a speed of 5 min;

(2) exposure of nucleic acids: dripping 0.2mol/L HCL on the tissue slices, and standing for 20min at room temperature; absorbing HCL, dripping Triton X-100, and standing at room temperature for 15 min; sucking out Triton X-100, and soaking in PBS solution for 5 min; dripping 5mmol/L proteinase K, standing at room temperature for 20min, and soaking in PBS solution for 5 min;

(3) and (3) sealing: dropping about 200 μ l of Blocking Buffer on the tissue slice, placing in a wet box, and sealing in a constant temperature box at 55 ℃ for 2 hours;

(4) preparing a probe: when the blocking is finished, diluting the probe and 25% Hybridization Buffer according to a ratio of 1:100, uniformly mixing, denaturing at 88 ℃ for 3min, and balancing at 37 ℃ for 5 min;

(5) and (3) hybridization: after sealing, sucking out Blocking Buffer, dripping 15-30 μ l of balanced probe, covering a cover glass, sealing the chip by Rubber Cement, and hybridizing at 37 ℃ for 48 hours;

(6) washing and dewatering: washing Buffer (10 ×) in distilled water at 1: 9, uniformly mixing to prepare working solution, removing Rubber comment, putting the glass slide into Washing Buffer I working solution, automatically dropping a cover glass after 5min, moving the glass slide to new Washing Buffer I working solution (preheated to 60 ℃), Washing for 2min, moving to room-temperature Washing Buffer II working solution, and Washing for 15 min; sequentially immersing the specimen in 70%, 85% and 100% ethanol, dehydrating for 2min, and drying at room temperature;

(7) sealing: 20 μ l of DAPI Anti-fade solution was added dropwise, covered with a cover slip, mounted with nail polish, left to stand in the dark for 15min, and observed under a confocal microscope.

The paraffin sections of examples 2 to 4 were all paraffin sections of the human brain glioma specimen described above.

The results show that: referring to FIG. 6(A) and FIG. 6(B) for bacterial LPS and LTA immunohistochemical staining of human glioma serial sections; FIG. 6(C) and FIG. 6(D) for bacterial LPS and LTA immunohistochemical staining of C57BL/6 mouse brain tissue serial sections (negative control), respectively; FIG. 6(E) and FIG. 6(F) for bacterial LPS and LTA immunohistochemical staining of C57BL/6 mouse intestine tissue serial sections (positive control)), example 2(IHC) suggests that bacterial LPS positive signals were detected in 3 samples of brain glioma and exhibited similar spatial distribution; bacterial LTA was not detected, which is consistent with previous reports, and no LPS and LTA positive signals were observed in the negative control samples, and both LPS and LTA positive signals were detected in the positive control samples.

Referring to fig. 7(a), fig. 7(B) and fig. 7(C) for bacterial LPS immunofluorescence staining of serial sections of human brain glioma, C57BL/6 mouse brain tissue (negative control) and small intestine tissue (positive control), respectively), example 3(IF) was the same antibody as example 1, and LPS was observed in all 3 glioma samples on thin paraffin sections, and was normally localized intracellularly in tumor tissue, while no LPS positive signal was observed in the negative control sample, and the same LPS positive signal was observed in the positive control sample, which laterally verified the effectiveness and specificity of the antibody, and added new evidence for the experimental result of gram-negative bacteria in glioma.

Referring to FIG. 8(A) and FIG. 8(B) for bacterial 16s rRNA labeling of continuous sections of human brain glioma with positive probe results on the left and negative probe results on the right, respectively; FIG. 8(C) and FIG. 8(D) for bacterial 16s rRNA labeling of continuous sections of C57BL/6 mouse brain tissue with positive probe results on the left and negative probe results on the right (negative control), respectively; FIG. 8(E) and FIG. 8(F) for bacterial 16s rRNA labeling of continuous sections of C57BL/6 mouse small intestine tissue with positive probe results on the left and negative probe results on the right (positive control)), example 4(FISH) suggests that bacterial 16s rRNA can be detected in human brain glioma samples and distributed beside the cell nucleus, while no bacterial 16s rRNA positive signal was observed in the negative control samples, the same 16s rRNA positive signal was observed in the positive control samples.

In conclusion, compared with the traditional histology method, the method combining the tissue transparency method and the traditional histology method can break the limitation (several microns) on the thickness of the tissue sample, so that the potential pollution on the surface of the thin slice is avoided, and more reliable results are obtained; the combined method of the invention adopts a chemical reagent quenching method to eliminate the interference of spontaneous fluorescent substances in tissues as much as possible, reduces false positive and further enhances the accuracy of the experiment. The prepared tissue can be imaged by a laser scanning microscope and a multiphoton laser scanning microscope, and three-dimensional reconstruction and analysis are carried out, so that the spatial distribution condition of bacteria in the tumor tissue and the overall appearance of the bacteria in a tumor microenvironment can be visually shown from a three-dimensional angle, and a foundation is laid for researching the interaction relationship between the tumor cells and the microorganisms of a host in the future.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of a combination of a tissue transparency method and a histological method for the detection of bacteria in tumors.

2. The use of claim 1, wherein the tissue clearing method comprises a hydrophobic clearing method or a hydrophilic clearing method.

3. The use of claim 1, wherein the histological method comprises one of immunofluorescent labeling, immunoenzymatic labeling, and fluorescent in situ hybridization labeling.

4. The use according to claim 1, wherein the tissue slices used in the detection of bacteria in tumours have a thickness of 400 to 600 μm.

5. The use of claim 4, wherein the tissue slices have a thickness of 500 μm.

6. The use of any one of claims 1 to 4, wherein the tumor comprises at least one of human brain glioma, breast cancer, pancreatic cancer, melanoma.

7. A tissue transparency method and an immunofluorescence labeling method are combined to be applied to a detection method of bacteria in tumors, and the detection method is characterized by comprising the following steps:

8. The detection method of claim 7, wherein the ratio of the primary antibody to the secondary antibody to the diluent in step 2) is 1:100, and the volume ratio of the tissue section to the diluent is 1:2 to 1: 3.

9. The assay of claim 7 wherein the primary antibody in step 2) is a monoclonal mouse lipopolysaccharide antibody and the secondary antibody is an donkey anti-mouse IgG antibody conjugated to Alexa Fluor Plus 594.

10. The assay of claim 7 wherein the primary and secondary antibodies are added in step 2) and incubated for at least 2 days.