CN115855894B - Probe and kit for identifying nucleolus and nucleolus labeling method - Google Patents

Abstract

The application relates to the technical field of molecular marker recognition, in particular to a nucleolus recognition probe, a nucleolus recognition kit and a nucleolus marking method. The nucleolus-recognizing probe provided by the application comprises a nucleotide sequence shown as GGXGGG, and the probe has a length of at least 20 nucleotides; the specific probe can be specifically combined with various nucleolin proteins, so that the nucleolin can be specifically marked, the nucleolin can be specifically marked and detected by the probe, the cost is low, the nucleolin phase separation structure can be well reflected, and the three-dimensional structure of the nucleolin can be reflected by an image obtained by imaging after marking, so that the probe has a good application prospect.

Description

Probe and kit for identifying nucleolus and nucleolus labeling method

Technical Field

The application belongs to the technical field of molecular marker recognition, and particularly relates to a nucleolus recognition probe, a nucleolus recognition kit and a nucleolus marking method.

Background

Nucleolus (nucleolus), an important organelle present in the nucleus, is the most prominent structure in the internuclear nucleus, and is known for its role in ribosome production, as the primary site of rRNA transcription and ribosome assembly. Since the production of ribosomes regulates overall metabolic activity in cells, nucleolus function is closely related to vital activities, such as cell cycle regulation and stress response. The morphology of nucleoli may reflect, in part, the identity of cells, the growth and metabolic status of cells, and abnormalities in the size and number of nucleoli often occur in cancer cells and are used as diagnostic tools for cancer. In addition, the nucleolus exhibits a membraneless phase separation structure that is different from the surrounding nucleoplasm. Studies have shown that liquid-liquid phase separation is the primary mechanism of nucleolar structure formation in eukaryotic cells. This phase separation phenomenon is thought to be widely present in many events within the cell, participating in important functions; the study of phase separation allows us to explain many of the previously difficult to understand phenomena. As the largest phase separation structure in the cell, research on the formation and organization of nucleoli can provide important information for the occurrence and structure of phase separation at other parts in the cell.

Currently, the nucleolus can be structurally divided into three layers, which are immiscible, including a Fibrous Center (FC), a dense fibril fraction (Dense Fibrillar Component, DFC) and a particulate fraction (Granular Component, GC), according to different characteristics observed under an electron microscope. The Fiber Center (FC) consists mainly of rDNA encoding ribosomal RNA surrounded by dense fibril components. Precursor rRNA transcribed at the fiber center boundary can be further processed into rRNA at dense fibril fraction layers. Finally, rRNA binds to ribonucleoprotein in the particle component layer to form ribosomal subunits. Since the three-layer structure of the nucleus can only be directly observed by electron microscopy and its exact boundaries are difficult to identify by contrast microscopy, investigation of its morphology usually requires reliance on fluorescent protein labeling. To date, a few targets for labeling nucleoli include: RNA polymerase I and upstream binding factor UBF for labeling fiber center, etc.; nucleolin, fibrillin, etc. for labeling dense fibril component layers; nucleophosmin and nucleolar frizzled phosphoprotein 1 for labeling the particle component layers, and the like. Labeling is typically performed using protein antibodies.

However, nucleoli contains more than 1300 proteins, although nucleoli is a relatively stable structure during the interphase of the cell cycle, it breaks down rapidly during cell division and most proteins shuttle between nucleoli and nucleoplasm. Thus, targeting one or a few proteins to label the nucleolus is difficult to exhibit the morphological structure of the nucleolus with sufficient accuracy, particularly during the cell division phase. In addition, the method of labeling newly synthesized RNA in nucleolus and super-resolution imaging nucleolus by means of fluorescent dye coupled 2 '-deoxyuridine-5' -triphosphate tetrasodium salt (2 '-deoxyuridine 5' -triphosphates, dUTP) or 5-Ethynyl-2'-deoxyuridine (5-ethyl-2' -deoxyuridine, edU) is relatively complex, time-consuming and costly.

Disclosure of Invention

The application aims to provide a probe, a kit and a nucleolus marking method for identifying nucleolus, which aim to solve the technical problem of how to simply, accurately and economically mark nucleolus.

In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:

in a first aspect, the present application provides a probe for nucleolus recognition, the probe is at least one of fifteen probes with nucleotide sequences shown in SEQ ID nos. 1 to 15, one end of the nucleotide sequence of the probe is connected with a fluorescent dye or a non-fluorescent substance generating fluorescence through chemical reaction, and the nucleotide sequences of the fifteen probes shown in SEQ ID nos. 1 to 15 specifically are:

the nucleotide sequence of SEQ ID NO.1 is from 5 to 3: TCGACGAATTGTATGGCCGGGCCGGGGGGCGGGGGGCCGGG; the nucleotide sequence of SEQ ID NO.2 is from 5 to 3: CGGTATCGATGCGAGGCCGGGCCGGGGGGCGGGGGGCCGGG; the nucleotide sequence of SEQ ID NO.3 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGGGTGGGTGGGTGGGTGGGTGGGT GGGCCGACG; the nucleotide sequence of SEQ ID NO.4 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGTGGGTGGGTGGGTGGGTGGGTGG GCCGACG; the nucleotide sequence of SEQ ID NO.5 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGTGGGTGGGTGGGTGGGTGGGCCG ACG; the nucleotide sequence of SEQ ID NO.6 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGGCGGGCGGGCGGGCGGGCGGGCG GGGCCGACG; the nucleotide sequence of SEQ ID NO.7 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCCGGGCGGGCGGGCGGGCGGGCGGG GCCGACG; the nucleotide sequence of SEQ ID NO.8 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCCGGGCGGGCGGGCGGGCGGGGCCG ACG; the nucleotide sequence of SEQ ID NO.9 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGGCGGGGTGGCGGGGTGGCGGGGT GGGCCGACG; the nucleotide sequence of SEQ ID NO.10 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGGGTGGCGGGGTGGCGGGGTGGCG GGGCCGACG; the nucleotide sequence of SEQ ID NO.11 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGTGGCGGGGTGGCGGGGTGGCGGG GCCGACG; the nucleotide sequence of SEQ ID NO.12 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCCGGGGTGGCGGGGTGGCGGGGTGG GCCGACG; the nucleotide sequence of SEQ ID NO.13 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGTGGCGGGGTGGCGGGGTGGGCCG ACG; the nucleotide sequence of SEQ ID NO.14 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCCGGGGTGGCGGGGTGGCGGGGCCG ACG; the nucleotide sequence of SEQ ID NO.15 is from 5 to 3: CCGGGCCGGGGGGCGGGGGGCCGGG.

In a second aspect, the present application provides a kit for identifying nucleoli, the kit comprising a probe as provided herein.

In one embodiment, the kit further comprises: cell fixing solution, cell membrane rupture solution, probe buffer solution and sealing buffer solution.

In one embodiment, the cell fixative is selected from the group consisting of aldehyde reagents; and/or the number of the groups of groups,

the cell rupture fluid is selected from solutions comprising nonionic detergents; and/or the number of the groups of groups,

the blocking buffer is selected from deoxyribonucleic acid mixture; and/or the number of the groups of groups,

the probe buffer is selected from sodium citrate buffer.

In a third aspect, the present application provides a method of marking a nucleolus, comprising the steps of:

fixing the cells to be marked on the climbing sheet by using a cell fixing liquid;

performing membrane rupture treatment on cells fixed on the climbing sheet by using a cell membrane rupture liquid, cleaning the climbing sheet, and adding a sealing buffer solution for performing first incubation treatment;

after dissolving the probe in a probe buffer solution, adding the probe to the surface of the climbing plate subjected to the first incubation treatment, and performing a second incubation treatment;

and (3) cleaning the climbing sheet after the second incubation treatment, sealing the climbing sheet with an imaging reagent, and then imaging.

In one embodiment, the cell fixative is selected from the group consisting of aldehyde reagents; and/or the number of the groups of groups,

the cell rupture fluid is selected from solutions comprising nonionic detergents; and/or the number of the groups of groups,

the blocking buffer is selected from deoxyribonucleic acid mixture; and/or the number of the groups of groups,

the probe buffer is selected from sodium citrate buffer.

In one embodiment, the temperature of the first incubation treatment is 15-24 ℃ for 60 minutes to 24 hours; and/or the number of the groups of groups,

the temperature of the second incubation treatment is 15-24 ℃ and the time is 10-36 hours.

The nucleolus-recognizing probe provided in the first aspect of the present application contains a specific nucleotide sequence shown in GGXGGG, and the specific oligonucleotide probe can be combined with various nucleolin, so that the nucleolin can be specifically marked in cells. The probe is used for carrying out specific marking detection on nucleolus, so that the cost is low, the nucleolus phase separation structure can be well reflected, and an image obtained by imaging after marking can reflect the three-dimensional structure of nucleolus, thereby having good application prospect.

The kit for identifying nucleolus provided by the second aspect of the application comprises the probe for identifying nucleolus, and based on the characteristics of the probe, the kit can be used for carrying out specific marking detection on nucleolus, so that the kit is low in cost, the nucleolus phase separation structure can be well reflected, the three-dimensional structure of nucleolus can be reflected by an image obtained by imaging after marking, and the kit has good application prospect.

According to the nucleolus marking method provided by the third aspect of the application, after fixing cells to be marked on a climbing sheet, rupture of membranes and closed incubation treatment, adding a special probe for incubation marking; the nucleolus can be specifically marked and detected based on the probe, so that the nucleolus marking method provided by the application can well reflect the nucleolus phase separation structure, and an image obtained by imaging after marking can reflect the nucleolus three-dimensional structure, and has good image resolution.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing a comparison of the imaging of probes and prior art labeled nucleolin provided in the examples of the present application.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects to be solved by the present application more clear, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.

In this application, the term "and/or" describes an association relationship of an association object, which means that there may be three relationships, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.

In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s).

It should be understood that, in various embodiments of the present application, the sequence number of each process does not mean that the sequence of execution is sequential, and some or all of the steps may be executed in parallel or sequentially, where the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the present application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.

In a first aspect, the present embodiment provides a nucleolus-recognizing probe, wherein the probe is at least one of fifteen probes with nucleotide sequences shown in SEQ ID No. 1-15.

The application proves that the oligonucleotide probe for recognizing nucleolus can be combined with a plurality of Nucleolin proteins, particularly Nucleolin proteins are combined with Nucleolin/RPL27/Ddx-27, and the Nucleolin proteins are positioned in DFC and GC subdomains of nucleolus, so that the probe provided by the application can specifically mark nucleolus in cells, and the nucleolus is specifically marked and detected by the probe, so that the cost is low, the separation structure of nucleolus can be well reflected, and the three-dimensional structure of nucleolus can be reflected by an image obtained by imaging after marking.

Specifically, the probes provided in the examples herein are capable of specifically labeling intracellular nucleoli and obtaining ultra-high resolution nucleoli phase separation by super-resolution imaging (STORM). The probe of the embodiment of the application has the following advantages: (1) The oligonucleotide probe is simple to prepare, low in cost, cheap and convenient; (2) The oligonucleotide probe is much smaller in size than the antibody, thus reflecting nucleolus phase separation structure more accurately; (3) Multiple nucleolin proteins can be identified simultaneously, and are more Robust (Robust). The nucleolus image obtained by the super-resolution imaging through the probe marking can well reflect the three-dimensional structure of nucleolus, and exceeds the resolution of the image obtained by the existing imaging method.

In one embodiment, the nucleotide sequence shown by GGXGGG may be GGCGGG; in particular, one or more nucleotide sequences shown as GGXGGG may be included to better specifically label the nucleolus of a cell, such as the nucleolus of a mammalian cell.

In one embodiment, the probe provided herein may be 20 to 60 nucleotides in length, specifically including: at least one of fifteen probes with nucleotide sequences shown in SEQ ID NO. 1-15. Specifically, one of them may be used, or two or more of them may be used in combination. The probe can be combined with nucleolin Nucleolin, RPL, ddx-27, and is not combined with Nucleosppin/B23 or Fibrilarin through experiments, so that the probe can well identify Nucleolin, RPL, ddx-27 parts of nucleolus.

In addition, the present application makes an alignment by selecting a probe not containing a GGXGGG sequence, for example: TCGACGAATTGTATGGCCAAACCAAAAAACAAAAAACCAAA; it was found experimentally that the probe sequence could not bind to nucleolus.

Fifteen probes shown in SEQ ID NOS.1-15 have target hybridization regions, can specifically identify the areas of the cell nucleolus Nucleolin, RPL and Ddx-27, and can realize nucleolus imaging by combining markers which generate fluorescent effects.

In one embodiment, the probe is attached to a label capable of producing a fluorescent effect, e.g., the label is selected from the group consisting of fluorescent dyes or non-fluorescent materials that produce fluorescence by chemical reaction, any fluorescent dye attached may emit light directly, and non-fluorescent material molecules attached to produce a fluorescent effect by chemical reaction may emit light indirectly. Thus, the present application achieves imaging display by binding of the above-described labels capable of generating fluorescence.

Specifically, the label may be directly or indirectly attached to one end of the nucleotide sequence of the probe. That is, the probe may be directly or indirectly attached to the label (e.g., a label probe is attached to the probe, and the label probe is attached to a fluorescent dye or a non-fluorescent substance that produces fluorescence by a chemical reaction), which are all within the scope of our embodiments, and the label may be attached to the 3 'end of the probe or to the 5' end of the probe. In a preferred embodiment of the present application, imaging of the label is achieved by direct attachment of fluorescent dyes to the probe. Any fluorescent dye commercially available is possible, including Alexa Fluor 647.

In a second aspect, embodiments of the present application provide a kit for identifying nucleoli, the kit comprising a probe as provided in the embodiments of the present application.

The kit for identifying nucleolus provided by the embodiment of the application comprises the probe for identifying nucleolus specific to the embodiment of the application, and based on the specific of the probe, the kit of the embodiment of the application can be used for carrying out specific marking detection on nucleolus, so that the kit is low in cost, the separation structure of nucleolus can be well reflected, the three-dimensional structure of nucleolus can be reflected by an image obtained by imaging after marking, and the kit has good application prospect.

In one embodiment, the kit further comprises: cell fixing solution, cell membrane rupture solution, blocking buffer solution and probe buffer solution. The cell fixing liquid is used for fixing the cells to be identified; the cell rupture fluid is used for rupture of the cell membrane so as to facilitate probe identification; the sealing buffer solution is used for quick and effective sealing; the probe buffer is used to solubilize the probe and wash the slide. The components of the kit are packaged separately, and are matched together for use to identify nucleolus.

Further, the cell fixing liquid is selected from aldehyde reagents such as paraformaldehyde solution, and paraformaldehyde solution is used as the cell fixing liquid, so that the cells to be marked can be well fixed on the climbing plate. The cell rupture fluid is selected from solutions containing nonionic detergents, such as Triton-X100 solution, which is good at rupturing the cell membrane. The sealing buffer solution is selected from deoxyribonucleic acid mixed solution, and has good sealing effect. The probe buffer solution is selected from sodium citrate (SSC) buffer solution, and the buffer solution has good cleaning effect.

A third aspect of embodiments of the present application provides a method of marking a nucleolus, comprising the steps of:

s01: fixing the cells to be marked on the climbing sheet by using a cell fixing liquid;

s02: performing membrane rupture treatment on cells fixed on the climbing sheet by using a cell membrane rupture liquid, cleaning the climbing sheet, and adding a sealing buffer solution for performing first incubation treatment;

s03: after dissolving the probe in a probe buffer solution, adding the probe to the surface of the climbing plate subjected to the first incubation treatment, and performing a second incubation treatment;

s04: and (3) cleaning the climbing sheet after the second incubation treatment, sealing the climbing sheet with an imaging reagent, and then imaging.

According to the nucleolus marking method provided by the embodiment of the application, after the cells to be marked are fixed on the climbing sheet, membrane rupture and closed incubation treatment, a special probe is added for incubation marking; the nucleolus can be specifically marked and detected based on the probe, so that the nucleolus marking method provided by the application can well reflect the nucleolus phase separation structure, and an image obtained by imaging after marking can reflect the nucleolus three-dimensional structure, and has good image resolution.

In step S01, the cells to be labeled are nucleolus-containing cells, and may be mammalian cells.

In one embodiment, the cell fixative is selected from aldehyde reagents, such as paraformaldehyde solution. Adding 3.5-4.5% paraformaldehyde solution onto the cell climbing sheet to be marked, and standing for 10-20 min to fix the cells. Then, the probe buffer was washed twice.

In step S02, the cell disruption solution is selected from solutions comprising nonionic detergents, such as Triton-X100 solution. Adding 0.1-0.3% Triton-X100 cell rupture liquid to the surface of the fixed cells on the climbing sheet, and standing for 5-15 min to rupture the fixed cells. Then, the probe buffer was used for washing.

In one embodiment, the blocking buffer is selected from the group consisting of a mixture of deoxyribonucleic acids. Specifically, adding a sealing buffer solution on the climbing slices after membrane rupture and cleaning, and performing first incubation treatment; the temperature of the first incubation treatment is 15-24 ℃ and the time is 60 min-24 h; the blocking effect is very good under the condition.

In step S03, the probe used is a probe specifically recognizing nucleolus in the examples of the present application. After the probe is dissolved in the probe buffer solution, the probe is added to the climbing sheet subjected to the first incubation treatment to perform the second incubation treatment, wherein the temperature of the second incubation treatment is 15-24 ℃ and the time is 10-36 h. Under these conditions the probe recognizes the bound nucleolus well.

In step S04, the second incubated slide is added to the probe buffer, immersed and washed, then sealed with an imaging reagent (e.g., a reagent containing an anti-quenching component or a buffer reagent required for STORM imaging), and then subjected to microscopic imaging.

The nucleolus-recognizing probe and the nucleolus labeling method using the probe provided by the embodiment of the application can be used as a very useful molecular tool in component medical diagnosis and cell biology research.

The following description is made with reference to specific embodiments.

Example 1

A nucleolus-recognizing probe selected from one of fifteen probes having nucleotide sequences shown in SEQ ID nos. 1 to 15, specifically shown in table 1:

TABLE 1

Code	Sequence number	Nucleotide sequence (5- & gt 3 terminal)
			M1	SEQ ID NO.1	TCGACGAATTGTATGGCCGGGCCGGGGGGCGGGGGGCCGGG
M2	SEQ ID NO.2	CGGTATCGATGCGAGGCCGGGCCGGGGGGCGGGGGGCCGGG
			M3	SEQ ID NO.3	CTCGACGAATTGTATGGCCGTCGGCGGGTGGGTGGGTGGGTGGGTGGGTGGGCCGACG
M4	SEQ ID NO.4	CTCGACGAATTGTATGGCCGTCGGCGTGGGTGGGTGGGTGGGTGGGTGGGCCGACG
			M5	SEQ ID NO.5	CTCGACGAATTGTATGGCCGTCGGCGTGGGTGGGTGGGTGGGTGGGCCGACG
M6	SEQ ID NO.6	CTCGACGAATTGTATGGCCGTCGGCGGCGGGCGGGCGGGCGGGCGGGCGGGGCCGACG
			M7	SEQ ID NO.7	CTCGACGAATTGTATGGCCGTCGGCCGGGCGGGCGGGCGGGCGGGCGGGGCCGACG
M8	SEQ ID NO.8	CTCGACGAATTGTATGGCCGTCGGCCGGGCGGGCGGGCGGGCGGGGCCGACG
			M9	SEQ ID NO.9	CTCGACGAATTGTATGGCCGTCGGCGGCGGGGTGGCGGGGTGGCGGGGTGGGCCGACG
M10	SEQ ID NO.10	CTCGACGAATTGTATGGCCGTCGGCGGGTGGCGGGGTGGCGGGGTGGCGGGGCCGACG
			M11	SEQ ID NO.11	CTCGACGAATTGTATGGCCGTCGGCGTGGCGGGGTGGCGGGGTGGCGGGGCCGACG
M12	SEQ ID NO.12	CTCGACGAATTGTATGGCCGTCGGCCGGGGTGGCGGGGTGGCGGGGTGGGCCGACG
			M13	SEQ ID NO.13	CTCGACGAATTGTATGGCCGTCGGCGTGGCGGGGTGGCGGGGTGGGCCGACG
M14	SEQ ID NO.14	CTCGACGAATTGTATGGCCGTCGGCCGGGGTGGCGGGGTGGCGGGGCCGACG
			M15	SEQ ID NO.15	CCGGGCCGGGGGGCGGGGGGCCGGG

The 3 'or 5' end of the probe is connected with fluorescent dye or a molecule capable of generating fluorescent effect.

Example 2

A kit for labeling a recognized nucleolus comprising the following components in separate packages:

(1) The probe of example 1;

(2) Cell fixative: 4% paraformaldehyde-PBS solution; paraformaldehyde, P6148, sigma-Aldrich; PBS buffer, SH30256.02, cytova.

(3) Cell rupture fluid: 0.2% PBS-Triton-X100 solution; triton X-100:9036-19-5, merck.

(4) Blocking buffer: a DNA mixture (Blocking Reagent, cat# 1109617671, roche) 15mg/ml Blocking Reagent was dissolved in 2 Xprobe buffer.

(5) 10 Xprobe buffer, SSC buffer (100 mL): naCl (378860, sigma-Aldrich) 8.769g,Sodium citrate tribasic dihydrate (6132-04-3, merck) 5.02g.

Example 3

A nucleolus labeling method using the kit of example 2 (specifically using the probe shown in SEQ ID NO.1, and having a fluorescent dye Alexa Fluor 647 attached to the 3' end of the probe) comprises the steps of:

(1) E14 cells (129 mouse ES cells) were fixed on a slide with 4% paraformaldehyde solution for 15 minutes, and then washed twice with PBS buffer;

(2) Cells fixed on the slide were treated with 0.2% PBS-Triton-X100 solution for 10min, then blotted off, rinsed with 80uL 2 XSSC buffer, and soaked in 1ml 2 XSSC buffer for 10 min;

(3) Adding a blocking buffer (blocking buffer), and incubating at 20 ℃ for 1 hour;

(3) After the probe shown in SEQ ID NO.1 of example 1 is prepared into a solution, the solution is added to a climbing plate and incubated for 12 hours at 20 ℃;

(4) Sucking the solution on the climbing plate, placing the climbing plate on a 24-hole plate, adding 1ml of 2 XSSC buffer solution, soaking for 10min, and repeating soaking for 3 times after sucking the SSC buffer solution;

(5) Sealing the chip with a reagent containing an anti-quenching component, preserving the sample, and observing through fluorescence microscopic imaging.

Meanwhile, respectively using the existing marker of the nucleolin: the methods of Fibrin (FB) and B23 were compared and the results are shown in fig. 1:

in fig. 1 a:

brightfield represents: white light;

M1-AF647 represents: the present example labels the display image with a probe that binds to Alexa Fluor 647;

FB-AF568 represents: the prior art Alexa Fluor 568 marks the nucleolin FB display image;

B23-AF488 represents: the prior art Alexa Fluor 488-labeled nucleolin B23 display image;

host represents: labeling the cell nucleus;

in fig. 1B:

M1-AF647 FB-AF568 represents: superposition amplification of M1-AF647 and FB-AF568 in A;

M1-AF 647B 23-AF 488: the superposition of M1-AF647 and B23-AF488 in A was amplified.

From the superposition analysis, it is known that: in the embodiment of the application, the nucleolin marked by the probe is different from the existing nucleolin marked by the probe, and the display image of the embodiment of the application can better reflect the three-dimensional structure of nucleoli and has good image resolution; in addition, other probes in Table 1 also had similar effects.

The foregoing description of the preferred embodiments of the present application is not intended to be limiting, but is intended to cover any and all modifications, equivalents, and alternatives falling within the spirit and principles of the present application.

Claims (7)

1. The probe for identifying nucleolus is characterized in that the probe is at least one of fifteen probes with nucleotide sequences shown as SEQ ID NO. 1-15, one end of the nucleotide sequence of the probe is connected with fluorescent dye or non-fluorescent substance generating fluorescence through chemical reaction, and the nucleotide sequences of the fifteen probes shown as SEQ ID NO. 1-15 are specifically:

the nucleotide sequence of SEQ ID NO.1 is from 5 to 3: TCGACGAATTGTATGGCCGGGCCGGGGGGCGGGGGGCCGGG; the nucleotide sequence of SEQ ID NO.2 is from 5 to 3: CGGTATCGATGCGAGGCCGGGCCGGGGGGCGGGGGGCCGGG; the nucleotide sequence of SEQ ID NO.3 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGGGTGGGTGGGTGGGTGGGTGGGT GGGCCGACG; the nucleotide sequence of SEQ ID NO.4 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGTGGGTGGGTGGGTGGGTGGGTGG GCCGACG; the nucleotide sequence of SEQ ID NO.5 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGTGGGTGGGTGGGTGGGTGGGCCG ACG; the nucleotide sequence of SEQ ID NO.6 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGGCGGGCGGGCGGGCGGGCGGGCG GGGCCGACG; the nucleotide sequence of SEQ ID NO.7 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCCGGGCGGGCGGGCGGGCGGGCGGG GCCGACG; the nucleotide sequence of SEQ ID NO.8 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCCGGGCGGGCGGGCGGGCGGGGCCG ACG; the nucleotide sequence of SEQ ID NO.9 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGGCGGGGTGGCGGGGTGGCGGGGT GGGCCGACG; the nucleotide sequence of SEQ ID NO.10 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGGGTGGCGGGGTGGCGGGGTGGCG GGGCCGACG; the nucleotide sequence of SEQ ID NO.11 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGTGGCGGGGTGGCGGGGTGGCGGG GCCGACG; the nucleotide sequence of SEQ ID NO.12 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCCGGGGTGGCGGGGTGGCGGGGTGG GCCGACG; the nucleotide sequence of SEQ ID NO.13 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCGTGGCGGGGTGGCGGGGTGGGCCG ACG; the nucleotide sequence of SEQ ID NO.14 is from 5 to 3: CTCGACGAATTGTATGGCCGTCGGCCGGGGTGGCGGGGTGGCGGGGCCG ACG; the nucleotide sequence of SEQ ID NO.15 is from 5 to 3: CCGGGCCGGGGGGCGGGGGGCCGGG.

2. A kit for identifying nucleoli, comprising the probe of claim 1.

3. The kit of claim 2, further comprising: cell fixing solution, cell membrane rupture solution, blocking buffer solution and probe buffer solution.

4. The kit of claim 3, wherein the cell fixative is selected from the group consisting of aldehyde reagents; and/or the number of the groups of groups,

the cell rupture fluid is selected from a solution comprising a nonionic detergent; and/or the number of the groups of groups,

the blocking buffer is selected from deoxyribonucleic acid mixed solution; and/or the number of the groups of groups,

the probe buffer is selected from sodium citrate buffer.

5. A method of marking a nucleolus, comprising the steps of:

fixing the cells to be marked on the climbing sheet by using a cell fixing liquid;

performing membrane rupture treatment on cells fixed on the climbing sheet by using a cell membrane rupture liquid, cleaning the climbing sheet, and adding a sealing buffer solution for performing first incubation treatment;

after the probe of claim 1 is dissolved in a probe buffer, the probe is added to the surface of the climbing plate subjected to the first incubation treatment, and a second incubation treatment is performed;

and (3) cleaning the climbing sheet after the second incubation treatment, sealing the climbing sheet with an imaging reagent, and then imaging.

6. A nucleolus marking method according to claim 5, wherein said cell fixative is selected from the group consisting of aldehyde reagents; and/or the number of the groups of groups,

the cell rupture fluid is selected from a solution comprising a nonionic detergent; and/or the number of the groups of groups,

the blocking buffer is selected from deoxyribonucleic acid mixed solution; and/or the number of the groups of groups,

the probe buffer is selected from sodium citrate buffer.

7. A method of marking a nucleolus as claimed in claim 5, wherein said first incubation treatment is carried out at a temperature of 15 to 24 ℃ for a period of 60 minutes to 24 hours; and/or the number of the groups of groups,

the temperature of the second incubation treatment is 15-24 ℃ and the time is 10-36 hours.