CN106198990A - A kind of tissue samples is carried out immune labeled method - Google Patents

Abstract

The invention discloses and a kind of tissue samples is carried out immune labeled method, specifically, the present invention (a) provides a labelling system, described labelling system to contain pending immune labeled tissue samples, for the probe being marked described tissue samples and buffer；(b) described labelling system is placed under electric field action it is marked process, so that described probe enters inside described tissue samples, thus described tissue samples is carried out immune labeled, it is thus achieved that through immune labeled tissue samples.The present invention can not only shorten probe and enter the time within tissue samples, moreover it is possible to keeps the integrity of organization internal, has great using value.

Description

A method for immunolabeling tissue samples

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for immunolabeling tissue samples.

Background technique

Studying the three-dimensional spatial structure of biomedical tissues at the cellular and subcellular scales is the basis for understanding their normal functions, and can also provide a basis for understanding the occurrence and development of organ diseases. Previous research on human and other animal tissues was mainly at the anatomical scale, while research at the cellular and subcellular scales was limited by the ability to analyze, and usually only the structural information of tissue slices could be studied. It is very time-consuming and labor-intensive to study tissue based on the 3D reconstruction technology of serial tissue slices.

In recent years, the rapid development of tissue clearing technology has made it possible for people to obtain high-resolution three-dimensional structures of complete biological tissues. At present, the most commonly used technology is CLARITY technology, which combines polymers formed by cross-linking of hydrogels with Biomolecules (proteins, DNA, etc.) in tissues are fixed, and biomolecules that strongly scatter light, such as cell membranes, are removed by using sodium dodecyl sulfate (SDS), which can ensure that the tissue structure is not damaged. Quickly transparentize biological tissues and realize the depth (~6mm) three-dimensional imaging of intact tissues. CLARITY technology was first applied to the transparent and structural research of mouse brain tissue, and gradually expanded to the main organs (kidney, liver, etc.) huge potential value.

However, when CLARITY technology is used to study the three-dimensional high-resolution structure of intact tissues, it takes a long time for labeled probes (especially antibodies) to reach the interior of the tissue from the surface of the intact tissue, which greatly hinders the promotion and application of this technology. . For example, for a 5 mm thick intact mouse brain sample, a full immunolabeling takes at least 1.5 months to complete.

Therefore, there is an urgent need in this field to develop a rapid immunolabeling method that can greatly shorten the immunofluorescent labeling time of tissue samples and maintain the internal integrity of the tissue.

Contents of the invention

The purpose of the present invention is to provide a rapid immunofluorescence labeling method that can greatly shorten the time for immunofluorescence labeling of tissue samples, and can also maintain the integrity of the interior of the tissue.

A first aspect of the present invention provides a method for immunolabeling a tissue sample, comprising the steps of:

(a) providing a labeling system comprising a tissue sample to be immunolabeled, a probe and a buffer for labeling the tissue sample; and

(b) placing the labeling system under the action of an electric field for labeling treatment, so that the probes enter the interior of the tissue sample, thereby immunolabeling the tissue sample, and obtaining an immunolabeled tissue sample.

In another preferred example, in step (b), the time for the labeling treatment is 1 min-5 h, preferably 20 min-1 h, more preferably 30 min.

In another preferred embodiment, in step (b), the temperature of the labeling treatment is 4-50°C, preferably 10-40°C, more preferably 37°C.

In another preferred example, the method further includes: (c) detecting the immunolabeled tissue sample.

In another preferred example, in step (c), the detection includes fluorescence detection.

In another preferred example, the electric field has the following characteristics: the voltage is 25V, the distance between electrodes is 2.2cm, and the electric field intensity is 11.3V/cm.

In another preferred example, the pH of the labeling system is 5-11.

In another preferred example, the probes include: antibodies and nucleic acid probes.

In another preferred example, the probe has a detectable marker.

In another preferred example, the detectable markers include: fluorophores, chromophores, and chemiluminescent groups.

In another preferred example, when the probe is an antibody, the pH of the labeling system is 5-11.

In another preferred example, the antibody is a monoclonal antibody.

In another preferred example, the isoelectric potential (PI) of the monoclonal antibody is 6.4-9.0.

In another preferred example, the tissue sample is a tissue sample expressing endogenous fluorescent protein.

In another preferred embodiment, the fluorescent protein is GFP protein.

In another preferred example, the tissue sample is brain tissue, stomach tissue, liver tissue, lung tissue or a combination thereof.

In another preferred embodiment, the tissue sample is derived from a mammal, a human, or a combination thereof.

In another preferred embodiment, the tissue sample is derived from mouse, rat, human, or a combination thereof.

In another preferred example, the tissue sample is a cleared sample.

In another preferred example, the sample is a sheet sample having a first main surface and a second main surface.

In another preferred example, the thickness of the sheet sample is 2-20mm, preferably 3-18mm, more preferably 5-10mm.

In another preferred example, the cross-sectional area of the sheet sample is 1-100 cm ² .

In another preferred example, the electric field is applied through electrodes located on the left and right sides or the upper and lower sides of the sample.

In another preferred example, the electric field is applied through electrodes located outside the first main surface and the second main surface of the sample.

In another preferred example, the time for the probe to enter the tissue sample is 20 min-1 h, preferably 30-50 min, more preferably 30-40 min.

In another preferred example, the time for the probe to enter the tissue sample is shortened by 800 times compared with traditional methods.

In another preferred example, the immunolabeling is immunofluorescent labeling.

In another preferred example, the method is a non-diagnostic and non-therapeutic in vitro method.

The second aspect of the present invention provides a device for immunolabeling tissue samples, the device comprising:

A container for placing the tissue sample; wherein, the container is used to hold a labeling system, the labeling system contains the tissue sample to be immunolabeled, a probe for labeling the tissue sample, and a buffer liquid;

A pair of electrodes for generating an electric field, wherein the pair of electrodes is located on the left and right sides or on the top and bottom sides of the tissue sample, so as to generate an electric field that drives the probe into the interior of the tissue sample; and

a power supply, the power supply is electrically connected to the pair of electrodes.

In another preferred example, the container is circular.

In another preferred example, the container has a diameter of 1-10 cm, preferably 3-4 cm, more preferably 3.5 cm.

In another preferred example, the diameter of the electrode is 0.1-1mm, preferably 0.2-0.8mm, more preferably 0.3-0.6mm; the length of the electrode is 2-15cm, preferably 4- 10cm, more preferably, 5-9cm.

The third aspect of the present invention provides a kit for immunolabeling tissue samples, the kit containing:

a first container containing a tissue sample to be immunolabeled;

a second container containing probes for labeling the tissue sample;

a third container, the container contains a buffer solution, and the pH of the buffer solution is 5-11;

The fourth container contains electrode plates and power plugs, forming an electric field with an electric field strength (V/cm) of 5-15.

A label or an instruction indicating that the kit is used for immunolabeling tissue samples.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the movement of IgG under the action of an electric field.

Figure 2 shows the results of fluorescent quantification of IgG in tissue gel complexes.

Figure 3 shows the YFP signal in Thy1-YFP mouse brain slices after electric field immunostaining.

Figure 4 shows the results of anti-YFP immunostaining of Thy1-YFP mouse brain slices subjected to electric field.

detailed description

After extensive and in-depth research, the inventor unexpectedly found for the first time that by adjusting the strength of the applied electric field, the probe can quickly enter the interior of the tissue sample, perform immunolabeling on the tissue sample, and maintain the integrity of the interior of the tissue. Specifically, When the applied electric field strength (V/cm) is 5-15, the probe can reach the inside of the tissue within 20min-1h, and perform immunolabeling on the tissue. The invention can not only shorten the time for the probe to enter the inside of the tissue sample, but also maintain the integrity of the inside of the tissue, and has great application value.

Immunolabeling of tissue samples

As used herein, the "immunolabeling of tissue samples" generally refers to a labeling method that uses antibodies to specifically bind antigens, which utilizes the specific binding properties of specific antibodies to isolate antigens, target antigens, and/or quantify antigens .

In the present invention, the "immunolabeling of tissue samples" refers to the use of probes to immunolabel the interior of tissue samples under the action of an external electric field. Compared with traditional methods, this method can not only accelerate the probe into Inside the tissue sample, it can also maintain the integrity of the inside of the tissue sample. Specifically, under the action of an external electric field, the probe can enter the tissue sample within 20min-1h, which greatly shortens the time for immunolabeling of complete transparent tissue. Compared with the traditional method of antibody molecular diffusion, this method The time can be shortened by 800 times.

The method for immunolabeling tissue samples provided by the present invention comprises the following steps:

(a) providing a labeling system comprising a tissue sample to be immunolabeled, a probe and a buffer for labeling the tissue sample;

(b) placing the labeling system under the action of an electric field for labeling treatment, so that the probes enter the interior of the tissue sample, thereby immunolabeling the tissue sample, and obtaining an immunolabeled tissue sample.

Device for Immunolabeling Tissue Samples

As used herein, the "device for immunolabeling tissue samples" includes:

A container for placing the tissue sample; wherein, the container is used to hold a labeling system, the labeling system contains the tissue sample to be immunolabeled, a probe for labeling the tissue sample, and a buffer liquid;

A pair of electrodes for generating an electric field, wherein the pair of electrodes is located on the left and right sides or on the top and bottom sides of the tissue sample, so as to generate an electric field that drives the probe into the interior of the tissue sample; and

a power supply, the power supply is electrically connected to the pair of electrodes.

Using the "device for immunolabeling tissue samples" of the present invention to perform immunolabeling on tissue samples can not only shorten the time for probes to enter the interior of the tissue samples, but also maintain the integrity of the interior of the tissue samples.

Kits for Immunolabeling of Tissue Samples

As used herein, the "kit for immunolabeling tissue samples" includes:

a first container containing a tissue sample to be immunolabeled;

a second container containing probes for labeling the tissue sample;

a third container, the container contains a buffer solution, and the pH of the buffer solution is 5-11;

The fourth container contains electrode plates and power plugs, forming an electric field with an electric field strength (V/cm) of 5-15.

A label or an instruction indicating that the kit is used for immunolabeling tissue samples.

The "kit for immunolabeling tissue samples" described in the present invention can not only quickly perform immunolabeling on the inside of tissue samples, but also is easy to carry.

The main advantages of the present invention include:

(1) Under the action of an external electric field, the probe can enter the interior of the tissue sample within 20 minutes to 1 hour, which greatly shortens the time for immunolabeling of complete cleared tissue. Compared with the traditional method of diffusion of antibody molecules, this method can shorten the time by 800 times.

(2) Under the action of an external electric field, the internal structure of the tissue sample remains intact.

(3) The present invention expands the application scope of the tissue transparency technology, and will be able to realize the research on the three-dimensional high-resolution structural information of large-scale biological tissues.

Below in conjunction with specific embodiment, further state the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate detailed conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Example 1 Preparation of brain tissue samples from cleared mice

Degreased mouse brain slices were trimmed into rectangles with a razor blade and mounted between two coverslips. Adjust the thickness of the blue butadiene to ensure that the blue butadiene can seal the upper and lower edges of the brain slices after the brain slices are loaded. Between the two coverslips, add a small amount of epoxy glue along the edge of the blu-tack.

Example 2 Configuring an electric field device for clearing mouse brain tissue samples

Put the prepared glass slide-brain slice combination into a petri dish, drill two small holes with a diameter of 1 mm on the lid of the 35 mm petri dish, and place two platinum electrodes with a diameter of 0.5 mm and a length of 8 cm (purchased from Sigma ) through the small hole, bend the electrode into a right angle, reinforce the electrode at the small hole with blue butadiene glue, add 2ml antibody diluent to the petri dish, put the lid on the petri dish, and load the petri dish on On the microscope stage, clamp the electrode with alligator clips and connect it to the power supply (KXN-6020D, ZHAOXIN).

Example 3 Detection results of electric field accelerated antibody entry into brain tissue samples

experimental method:

1) Dilute 6 μl of IgG (purchased from Molecular Probes Inc.) into 2000 μl of 0.1 M sodium borate buffer (pH 8.5).

2) Add the antibody dilution to the culture dish, and cover the electrode cover.

3) After finding the diffusion edge of the sample under the microscope, take pictures and record it.

4) After standing still for 30 minutes, take pictures again to record the diffusion of antibody molecules in the brain slices.

5) Turn on the power, adjust the voltage to 25V, take pictures and record after electrophoresis for 30 minutes.

6) Process pictures and extract information.

Experimental results:

(1) As shown in Figure 1-2, the results show that under the action of the electric field, the antibody filled the whole brain slice more uniformly. Compared with the control group without electric field, the antibody can fill the whole brain slice within 30 minutes .

(2) If the tissue depth at which the antibody concentration reaches 50% of the maximum concentration is defined as the antibody diffusion front, then within 30 minutes IgG diffused to a depth of 3.9mm inside the tissue. Therefore, the time required for IgG molecules to freely diffuse to the same diffusion front is approximately 800 times that of the case without an applied electric field.

Example 4 Immunostaining of Mouse Brain Tissue Samples

experimental method:

1) Mount the fat-free Thy1-YFP mouse brain slices.

2) 6 μl of IgG antibody (anti-GFP antibody, purchased from Molecular Probes Co., Ltd.) was diluted in 2000 μl of 0.1 M sodium borate buffer (pH 8.5), and slowly added to the culture dish.

3) Turn on the power, adjust the voltage to 25V, and perform electrophoresis for 30 minutes to help the antibody enter the tissue.

4) Turn off the power. Incubate for 90 minutes to allow the antibody and antigen to fully combine.

5) Turn on the power. Adjust the voltage to 25V, run electrophoresis for 30 minutes, and remove unbound antibodies inside the tissue.

6) Aspirate the antibody diluent in the petri dish, and replace it with 2ml of clean 0.1M boric acid buffer.

7) Imaging, observing the marking results.

Experimental results:

As shown in Figure 3-4, after 60 minutes of electrophoresis, the YFP signal in Thy1-YFP mouse brain slices is still well protected, and the YFP signal can well overlap with the antibody signal.

The results show that the external electric field can help the IgG antibody quickly enter the tissue sample and perform immunolabeling on the tissue interior.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A method for immunolabeling tissue samples, comprising the steps of: (a) providing a labeling system comprising a tissue sample to be immunolabeled, a probe and a buffer for labeling the tissue sample; and (b) placing the labeling system under the action of an electric field for labeling treatment, so that the probes enter the interior of the tissue sample, thereby immunolabeling the tissue sample, and obtaining an immunolabeled tissue sample. 2. The method according to claim 1, further comprising: (c) detecting the immunolabeled tissue sample. 3. The method according to claim 2, characterized in that, in step (c), said detection comprises fluorescence detection. 4. The method according to claim 1, characterized in that the pH of the labeling system is 5-11. 5. The method of claim 1, wherein the sample is a sheet sample having a first major surface and a second major surface. 6. The method according to claim 5, characterized in that, the cross-sectional area of the sheet sample is 1-100 cm ² . 7. The method according to claim 1, wherein the electric field is applied through electrodes located on the left and right sides or the upper and lower sides of the sample. 8. A device for immunolabeling tissue samples, characterized in that the device comprises: A container for placing the tissue sample; wherein, the container is used to hold a labeling system, the labeling system contains the tissue sample to be immunolabeled, a probe for labeling the tissue sample, and a buffer liquid; A pair of electrodes for generating an electric field, wherein the pair of electrodes is located on the left and right sides or on the top and bottom sides of the tissue sample, so as to generate an electric field that drives the probe into the interior of the tissue sample; and a power supply, the power supply is electrically connected to the pair of electrodes. 9. The device of claim 8, wherein the container has a diameter of 1-10 cm. 10. A kit for immunolabeling tissue samples, characterized in that the kit contains: a first container containing a tissue sample to be immunolabeled; a second container containing probes for labeling the tissue sample; a third container, the container contains a buffer solution, and the pH of the buffer solution is 5-11; The fourth container contains electrode plates and power plugs, forming an electric field with an electric field strength (V/cm) of 5-15. A label or an instruction indicating that the kit is used for immunolabeling tissue samples.