KR101161238B1

KR101161238B1 - Verification Method of nuclear antigen and Positioning Method by immunonucleochemistry

Info

Publication number: KR101161238B1
Application number: KR20100041374A
Authority: KR
Inventors: 문일수; 이현숙; 정은정
Original assignee: 동국대학교 경주캠퍼스 산학협력단
Priority date: 2010-05-03
Filing date: 2010-05-03
Publication date: 2012-07-02
Also published as: KR20110121876A

Abstract

The present invention relates to a method for verifying that an antigen is located in the nucleus of a cell. More specifically, the present invention relates to a method of verifying that antigens are present in a nucleus by directly applying a detergent to cells attached to a substrate to remove cell membranes, removing cytoplasm, and staining only nuclei by immunocytochemistry.
The present invention can be used to clarify simply and clearly whether the cell membrane lysate is directly processed to cells attached to the substrate whether a particular antigen of the cell is located in the nucleus or cytoplasm.

Description

Verification Method of nuclear antigen and Positioning Method by immunonucleochemistry}

The present invention relates to a method for verifying that an antigen is located in the nucleus of a cell. More specifically, the present invention relates to a method of verifying that antigens are present in a nucleus by directly applying a detergent to cells attached to a substrate to remove cell membranes, removing cytoplasm, and staining only nuclei by immunocytochemistry.

Accurate positioning of antigens in cells via immunohistochemistry (IHC) is essential for understanding the spatiotemporal expression of the antigen. Immunohistochemistry is a method of binding a primary antibody to a tissue antigen and attaching a secondary antibody to the primary antibody. In this way, the site or morphology of the expressed tissue can be identified, and the site of antigen expressed in the tissue can be identified. If Western blotting is a technical test that only knows whether it is expressed, immunohistochemistry is a kind of analytical test that observes with the naked eye exactly where the protein is expressed.

The first attempt at this method was made in the 1940s by Albert H. Coons and his colleagues (Coons et al., 1941, 1942). Since then, various IHC methods have been devised. For example, peroxidase enzymes can be linked to antibodies (Nakane and Pierce 1966; Avrameas and Uriel 1966) or alkaline phosphatase enzymes (Mason and Sammons 1978). Colloidal gold can be used for electron microscopy (Faulk and Taylor 1971).

In contrast to IHC using tissue sections, immunocytochemistry (ICC) is a method applied to isolated cells, which is a case of separation from cultured cells or tissues. Immunocytochemistry is an anatomical method used to identify specific molecules in specific cells. The principle is very simple. For example, to demonstrate that certain neurotransmitter molecules are synthesized in certain neurons, the neurotransmitter candidate molecules are chemically purely isolated and injected into the bloodstream of the animal to stimulate an immune response. Occasionally, candidate molecules and larger molecules are chemically combined to induce an immune response. One of the immune responses is the production of antibodies that bind tightly to specific positions of this substance molecule. These specific antibody molecules are recovered from blood samples of immunized animals and labeled with markers of the color that can be seen on the microscope. This labeled antibody is then applied to the brain tissue sections to bind the candidate molecule so that only cells containing the candidate molecule will be shown in a particular color. Immunocytochemistry can be used to locate cells with molecules (neurotransmitters and synthetases) that caused specific antibodies to be produced. Demonstrating that the neurotransmitter candidate and its synthetase are at the same axon end or contained within the neuron can satisfy the condition that the molecule is synthesized and present in a particular neuron.

The advantage of ICC is that the spatiotemporal location of antigens in cells can be known in more detail. However, if the organelles overlap in the z-axis, it is difficult to clearly identify the exact location of the antigen on the microscopic image. In this respect, there are more problems in identifying expression in the nucleus than in any intracellular organelles. Because the nucleus is a relatively large organelle, it overlaps with the surrounding cytoplasm. Therefore, the current method for determining whether an antigen is located in the nucleus or cytoplasm is by Western blotting by separating the nuclear fraction and the cytoplasmic fraction (McEwen et al., 1980; Garrido-Garc et al. , 2009). However, it is almost impossible to separate the two fractions without cross contamination from each other. Therefore, it is necessary to develop a technology for purely separating the nucleus.

The present inventors have come to devise a method for clarifying whether the antigen is located in the nucleus by separating the nucleus.

It is an object of the present invention to provide a method for verifying that an antigen is located in a cell. More specifically, the present invention relates to a method of verifying that antigens are present in a nucleus by directly applying a detergent to cells attached to a substrate to remove cell membranes, removing cytoplasm, and staining only nuclei by immunocytochemistry.

In order to achieve the object of the present invention as described above, the present invention

(a) treating the cells attached to the substrate with detergent to remove the cell membrane;

(b) removing the cytoplasm after removing the cell membrane;

(c) subjecting the nucleus of the cell from which cytoplasm has been removed to an immunostaining process;

It provides a method for verifying the nuclear antigen by immunocytochemistry comprising a.

Preferably, the substrate of step (a) is a coverslip.

The detergent of step (a) is a detergent for removing the cell membrane without damaging the nuclear membrane, but is not limited thereto, but NP-40 or Triton X-100 is preferred.

The concentration of the detergent of step (a) is preferably 0.1 ~ 1.0% (v / v), the treatment time of the detergent is preferably 20 minutes or less.

Cellular removal of step (b) is preferably washed with phosphate-buffered saline (PBS).

It is preferable to fix the cells before the immunostaining process of step (c), and the fixation of the cells is preferably paraformaldehyde / methanol.

The present invention also provides a nuclear antigen detected by the above method.

The present invention can be used to clarify whether the specific antigen of the cell is located in the nucleus or cytoplasm, directly treating the cell membrane lysate to the cells attached to the substrate.

Figure 1 shows a flow chart of the cell nuclear immunoassay method.
2 is a phase difference micrograph of rat hippocampal neurons at day 17 (DIV 17) of cell membrane removal and nuclei exposed by NP-40 treatment. (A) control group, (B) the experimental group treated with 0.5% NP-40 (v / v) for 5 minutes. Below is an enlarged picture of the square area, showing the border of the nerve cell nucleus (arrow), cytoplasmic residue (asterisk), the nucleus border of glial cells (white arrow), scale bar, and 20 μm.
3 shows cell membrane lysis according to NP-40 concentration. Hippocampal neurons of DIV 17 were phase contrast micrographs (AD) treated with 0.0, 0.1, 0.5, and 1.0% NP-40 for 5 minutes, respectively. Pictures were taken at the nuclear level on the z-axis to show the nucleus boundaries (arrows). Nucleus border (arrow), cell body residue (star) Large branching residue (white star), small branching (arrow head), scale bar, 20 μm.
4 shows cell membrane lysis according to NP-40 treatment time. Hippocampal neurons of DIV 17 were phase contrast micrographs (AD) treated with 0.5% NP-40 for 2, 5, 10, and 20 minutes, respectively. Pictures were taken at the nuclear level on the z-axis to show the nucleus boundaries (arrows). In B and D, after the NP-40 treatment, photographs of cells that had been washed and fixed were inserted. Nucleus rim (arrow), cell body residue (asterisk) large dendritic residue (white asterisk), scale bar, 20 μm.
5 shows cell membrane removal of cell membrane lysates prepared with Triton X-100. Hippocampal neurons of DIV 1 were phase contrast micrographs (5A-D) treated with 0.0, 0.1, 0.5, and 1.0% Triton X-100 for 5 minutes. Pictures were taken at the nuclear level on the z-axis to show the nucleus boundaries (arrows). Scale bar, 20 μm.
6 shows lysis of non-neuronal cell membranes. Cultured human embryonic kidney 392T (HEK392T) cells 0.1. Photographs treated with 0, 5 and 10 minutes with 0.5, and 1.0% NP-40 and washed cells (AC). Nuclear borders (arrows), cell body residues (asterisks), and large dendritic residues (white asterisks). Scale bar, 20 μm.
Figure 7 Isolation of the signal expressed in the nucleus part from intracellular expression by the method of the present invention. Cells of developmental stage 3 (axonal outgrowth) were immunostained with an antibody against N-acetylglucosamine kinase (NAGK). (A) control group, NP-40 was not treated, (B) experimental group. When treated with NP-40 by the method of the present invention (0.5%, 5 min), NAGK spots located in the nucleus were clearly separated from the strong signal (A) of the cytoplasm (B). Scale bar, 20 μm.

Hereinafter, the present invention will be described in detail.

The present invention,

(b) removing the cytoplasm after removing the cell membrane;

Using the cells attached to the substrate of step (a) may attach the nucleus to which the cell membrane and cytoplasm has been removed by treating the suspended cell with the substrate, but the nucleus itself has weak adhesion and detergent treatment and attachment time. This may lead to denaturation of substances in the nucleus, while the substrate may be a cell culture plate, a slide glass, coverslips, but is not limited thereto. (coverslip) is preferred. In more detail, in one embodiment of the present invention, a phosphate-buffered saline (phosphate-buffered) is placed on a slide glass and placed in ice water so that the cells are covered with a coverslip attached thereto. wash briefly with saline (PBS).

The method of removing the cell membrane in step (a) includes osmotic swelling or a method of treating a detergent, but is not limited thereto. It is preferable to remove the cell membrane using a detergent. Detergents remove cell membranes, such as SDS and CTBA, which are ionic detergents, and polyoxyethylene glycol, which are non-ionic degergents (e.g., Tween, Triton, NP-40, Brij-based), CHAPS, Glycosides (e.g. octyl-thio-glucoside, maltosides), bile acids (e.g. DOC, HEGAs), or phosphine oxides, including but not limited to efficient removal of cell membranes without damaging the nuclear membrane NP-40, Triton X-100, which do not interfere with interprotein or intraprotein binding, are preferred. Depending on the type and condition of the cells, the type and concentration of the detergent may be increased or decreased. The membrane removal method may be used for all cells isolated from cultured cells or tissues.

The concentration of the detergent of step (a) is preferably 0.1 ~ 1.0% (v / v). In one embodiment of the present invention, a lysis buffer containing NP-40 or Triton X-100 at various concentrations (0.1-1%, v / v) diluted with PBS was used. The treatment time of the detergent is variable depending on the concentration of the detergent. The treatment time is preferably 20 minutes or less at room temperature, but more preferably 2 to 5 minutes. If the treatment time exceeds 20 minutes, it is not preferable because intracellular metabolism and enzymatic activity occurs even during detergent treatment.

Cellular removal of step (b) is preferably washed with phosphate-buffered saline (PBS). It may include washing 2 to 4 times to remove the cellular components with phosphate buffered saline.

It is preferable to fix the cells before the immunostaining process of step (c), and the fixation of the cells is preferably paraformaldehyde / methanol (paraformaldehyde / methanol) method. The paraformaldehyde / methanol method was treated with 4% paraformaldehyde dissolved in PBS in an embodiment of the present invention for 10 minutes at room temperature and washed with water, then treated with -20 ° C. methanol for 20 minutes at -20 ° C. (Moon et al., 2007).

Immunostaining process of the nucleus of step (c) is H-PBS added with preblocking buffer (preblocking buffer) [5% normal goat serum, 0.05% Triton X-100 to the fixed cells in one embodiment of the present invention (450 mM NaCl, 20 mM phosphate buffer, pH 7.4)] and treated overnight at 4 ° C. (˜16 hours). The next day, the cells were washed three times with the TCM and the secondary antibodies [Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 647-conjugated goat anti-chicken IgG (diluted 1: 1,000 each) Add and process 1-2 hours at room temperature. After the treatment, the cover slip is washed three times with a total blocking buffer solution and three times with PBS at room temperature, placed on a glass slide for observation, and then observed under a microscope.

The present invention also provides a nuclear antigen detected by the above method. Nuclear antigens refer to antigens present in the nucleus of cells.

Hereinafter, the present invention will be described in detail through examples. The following examples are only illustrative of the present invention, and the scope of the present invention is not limited thereby.

< Example >

1. Preparation of cell membrane lysate

(One) NP Preparation of Cell Membrane Lysate Using -40

Cell membrane lysates can be prepared using conventional detergents. Surfact-Amps NP-40 (NP-40 10% (v / v) aqueous solution) from Pierce (Rockford, IL, USA) was diluted to 0.1, 0.5 and 1.0% (v / v) with PBS.

(2) Triton Preparation of Cell Membrane Lysate Using X-100

Surfact-Amps X-100 (Triton X-100 10% (v / v) aqueous solution) from Pierce (Rockford, IL, USA) was diluted to 0.1, 0.5 and 1.0% (v / v) with PBS.

The results using the membrane lysis solution are as follows.

2. Results

(One) NP Membrane Removal Efficiency of Membrane Lysis Agents Prepared with -40

The rats (murine) used in this example are Sprague-Dawley systems and are representative experimental animals used in experiments worldwide. Rat fetal cortical neurons on embryonic day 18 (E18) were bred by Brewer et al. (Brewer, GJ, Torricelli, JR, Evege, EK and Price, PJ, Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.J. Neurosci.Res., 35, 567-576, 1993). That is, on the 18th day of pregnancy, the fetus was taken from the uterus of the rat, the brain was dissected, and the hippocampus was separated and chopped. Tissues were treated with 0.25% (w / v) trypsin at 37 ° C. for 10 minutes, and the cells were dispersed by passing through the paste 6-6 times with a small Pasteur pipette with a small hole. . Were cultured in collected by centrifuging the dispersed cells, and then measuring the number of cells inoculated at a neuro bay added to well B27 medium (Invitrogen, _{USA) 5% CO 2/37 °} C culture tank. Treated for 5 minutes with a lysate (Example 1) prepared using 0.5% NP-40 (v / v) on day 17 in culture (DIV) 17 days (B, experimental group) or not treated ( A, control) A photo taken of the cells with a phase contrast microscope is shown in FIG. Cells of the experimental group treated with the membrane lysate had more dendrites dissolved than the cells of the control group, and the edge of the nerve cell nucleus (arrow) is clearly seen in the enlarged photograph of the square area. Also visible is the nucleus border (white arrow) of glial cells. However, the border of the nucleus is not clear in the cells of the control group. The rim of the nucleus is clearly visible because the cell membrane is lysed and the nucleus of the cell is taken out. Thus, this experimental example demonstrates that the cell membrane lysate prepared according to the present invention dissolves the cell membrane very efficiently.

(2) NP Membrane Lysis at -40 Concentration

Figure 5 shows a phase contrast microscope photograph of 5 minutes of DIV 17 hippocampal neurons cultured in the same manner as in (1), each treated with a membrane lysate (Example 1) prepared with 0.0, 0.1, 0.5, and 1.0% NP-40. It is shown to 3A-D, respectively. Pictures were taken at the nuclear level on the z-axis to show the nucleus boundaries (arrows). In 0.1, 0.5 and 1.0% NP-40 all nuclei are clearly visible (arrows). Therefore, this experimental example demonstrates that the cell membrane lysate prepared by the present invention dissolves the cell membrane very efficiently at various concentrations.

(3) NP Membrane Lysis with Treatment Time

Phase contrast micrographs of 2, 5, 10, and 20 minutes of DIV 17 hippocampal neurons cultured in the same manner as in (1) were respectively treated with a membrane lysate (Example 1) prepared with 0.5% NP-40. Shown in 4A-D. Pictures were taken at the nuclear level on the z-axis to show the nucleus boundaries (arrows). In B and D, after the NP-40 treatment, photographs of cells that had been washed and fixed were inserted. Even after two minutes, the nucleus edge (arrow) is clearly visible. Meanwhile, even after 20 minutes of treatment, the nucleus does not fall off and the cell membrane is removed and shows that it is well attached. Therefore, this experimental example demonstrates that the cell membrane lysate prepared by the present invention not only dissolves the cell membrane very efficiently at various treatment times, but also stably attaches the nucleus without falling off from the substrate.

(4) Triton Cell Membrane Removal Effect of Membrane Lysis Agents Prepared with X-100

Phase contrast micrographs of DIV 17 hippocampal neurons cultured in the same manner as in (1) above were treated with a membrane lysate (Example 2) prepared with 0.0, 0.1, 0.5, and 1.0% Triton X-100 for 5 minutes, respectively. 5A-D are shown respectively. Pictures were taken at the nuclear level on the z-axis to show the nucleus boundaries (arrows). In 0.1, 0.5 and 1.0% Triton X-100, the nucleus rim is clearly visible (arrow). Therefore, this experimental example demonstrates that the cell membrane lysate prepared by the present invention dissolves the cell membrane very efficiently in various detergents and concentrations.

(5) Non-neuronal cells ( non - neuronal cell Lysis of cell membrane

In order to demonstrate that the membrane lysate prepared in the present invention works efficiently in cells other than neurons, cultured human embryonic kidney 392T (HEK392T) cells were prepared in 0.1. The photographs treated with 0, 5 and 10 minutes with 0.5, and 1.0% NP-40 and washed cells are shown in FIGS. 6A-C. At various concentrations and treatment times, the edges of the nucleus are apparent (arrows). Therefore, this experimental example demonstrates that the cell membrane lysate prepared by the present invention dissolves cell membranes of various cells very efficiently.

(6) demonstration of the presence in the nucleus of the antigen

N-acetylglucosamine kinase (NAGK) is also expressed in the cytoplasm but strongly expressed in the form of spots in the nucleus. To show that strongly expressed spots are located in the nucleus rather than the cytoplasm, cells in development stage 3 (axonal outgrowth) were treated with 0.5% NP-40 membrane lysate for 5 minutes (experimental group) or untreated ( Control group) and immunostained with antibody to NAGK. In FIG. 7, it is impossible to distinguish whether the immunostaining signal is in the cytoplasm or the nucleus in the control group (A) not treated with NP-40. However, the treatment of NP-40 by the method of the present invention (B, experimental group) shows that the immunostaining signal is maintained even when the cell membrane is dissolved and all the cellular components are removed. This shows that the NAGK antigen is concentrated in spots in the nucleus. This example thus demonstrates that the method of the present invention can be used to clearly distinguish antigens of the cytoplasm and the nucleus.

In summary, the treatment of cells attached to the substrate with a detergent removes the cell membrane and efficiently removes the cytoplasmic components. The nucleus remains exposed and stably attached to the substrate. It turns out that it can clearly prove its position in.

Although embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. I can understand that. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims

(a) attaching the cells to the substrate;
(b) treating NP40 or Triton-X directly with the cells attached to the substrate to remove the cell membrane;
(c) removing the cytoplasm after removing the cell membrane; And
(d) subjecting the nucleus of the cell from which the cytoplasm has been removed to an immunostaining process; How to distinguish between cytoplasmic antigen and nuclear antigen by immunocytochemical method comprising a.

The method of claim 1,
And the substrate of step (a) is a coverslip.

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The method of claim 1,
NPb or Triton-X concentration of step (b) is characterized in that the 0.1 ~ 1.0% (v / v).

The method of claim 1,
The treatment time of NP40 or Triton-X of step (b) is characterized in that more than 2 minutes 20 minutes or less.

The method of claim 1,
The cytoplasmic removal of step (c) is characterized in that the washing with phosphate-buffered saline (phosphate-buffered saline, PBS).

The method of claim 1, further comprising fixing the cells prior to the immunostaining step (d).

8. The method of claim 7, wherein the cell is fixed by a paraformaldehyde / methanol method.

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