KR101486196B1 - Antibody tissue penetration acceleration technique using pressure - Google Patents

Abstract

The present invention relates to a method for coloring tissue, wherein an antigen and antibody reaction in tissue is performed under pressure. In the method according to the present invention, the tissue permeability of an antibody is increased so that an antigen and antibody reaction can be performed even in a deep part of tissue.

Description

[0001] The present invention relates to an antibody-tissue penetration accelerating technique using pressure,

The present invention relates to techniques for accelerating penetration of antibody tissue utilizing pressure.

Tissue staining method is a method of identifying a specific substance present in a tissue by using an antigen-antibody reaction, and is a method of staining which is frequently used in basic science research, diagnosis science.

The principle of tissue dyeing method is as follows. When the anti-A antibody (primary antibody) against the substance A present in the tissue is reacted with the tissue, the antibody reacts with the antigen A. However, even though an antigen-antibody conjugate is formed, it is difficult to observe under an optical microscope since the antibody is a kind of immunoglobulin. Therefore, a fluorescent dye or the like may be labeled on an antibody (secondary antibody), and an antigen-antibody reaction may be performed to confirm the localized region of the antigen. In such a tissue dyeing method, the antibody must penetrate into the tissue, and thus the depth of the tissue that can be observed depending on the tissue permeability of the antibody is determined (Non-Patent Documents 1 to 3).

However, even if the primary antibody is allowed to react with the tissue for a long time, for example, about 48 hours, the depth of the tissue that can be observed is about 40 탆 at the maximum.

In recent years, techniques for visualizing brain tissue (non-patent document 4) and imaging of deep tissue of tissue (SeeDB, non-patent document 5) have been developed. However, there is a time limitation that the brain tissue transparency technique requires about 30 days to dye the tissue. In addition, since SeeDB technology can only image to the place where immunological staining is done, there is a disadvantage that it can not be imaged unless the tissue deep part is dyed. That is, there are barriers to the time of the first antigen-antibody reaction and the tissue permeability of the antibody, and there is a difficulty in using the previously developed technique.

1. Coons AH. Et al., Proc Soc Exp Biol Med 1941; 47: 200-202. 2. J Exp Med 1950; 91: 1-13., Gen Cytochem Methods 1850: 1-13. 3. Gen Cytochem Methods 1958; 1: 399-422. 4. chung K. et al., Nature., 497 (7449): 332-7, 2013 5. Ke MT. Et al., Nat Neurosci., 16 (8): 1154-61., 2013

It is an object of the present invention to provide a method of staining tissue capable of enhancing the tissue permeability of an antibody and performing an antigen-antibody reaction to the deep portion of the tissue for a short time.

In the present invention, in tissue dyeing,

The present invention provides a method of staining a tissue comprising performing the reaction of an antigen in a tissue with an antibody under pressure.

In the method of the present invention, the antigen-antibody reaction can be performed to the deep part of the tissue by increasing the tissue permeability of the antibody.

In addition, the antigen-antibody reaction time can be shortened and the tissue can be imaged more quickly.

Further, by shortening the staining time and securing the depth of the observable tissue, it can be usefully used when it is combined with the brain tissue transparency technology and the SeeDB technology in the future.

1 is a schematic view of a kit including a hydrogel bed according to an embodiment of the present invention.
FIG. 2 is a view illustrating a procedure of performing a tissue dyeing method under a positive pressure condition according to an embodiment of the present invention.
FIG. 3 is a diagram illustrating a procedure of performing a tissue dyeing method under mixing conditions of negative pressure and positive pressure according to an embodiment of the present invention.
FIG. 4 is a photograph (A) of a tissue stained by the tissue staining method of Example 1 and Comparative Example, a graph (B) showing the depth of antibody penetration and fluorescence signal intensity, and a depth comparative graph )to be.
5 is a photograph (A) of a tissue stained by the tissue immuno-staining method of Example 2 and a comparative example, a graph of intensity change of a fluorescent signal as a result of staining, a graph (C) of a comparison of signal intensity sections capable of quantitative analysis, (D) is the Z axis depth comparison of the dyed tissue.
FIG. 6 is an imaging photograph (A) of a tissue stained by the tissue immunostaining method of Example 3 and a comparative example, and a Z axis depth comparison photograph (B) of the stained tissue.
Fig. 7 is a graph showing the intensity change of the fluorescent signal as a result of staining of the tissue as a result of the tissue immuno staining of Example 4 and Comparative Example 3 (A); and a photograph (C) of comparing the Z axis depth of the stained tissue.

In the present invention,

The present invention relates to a method of staining a tissue, which comprises performing a reaction between an antigen in a tissue and an antibody under pressure.

Hereinafter, the method for staining tissue according to the present invention will be described in more detail.

In the present invention, the reaction of the antigen in the tissue with the antibody can be carried out by specifically reacting the antibody to the antigen of the fixed tissue, and the reaction can be carried out under pressure. The reaction of the antigen in the tissue with the antibody can be referred to as an 'antigen-antibody reaction'.

In the antigen-antibody reaction, the tissue is not particularly limited as long as it can be used in the immuno-staining method. For example, cultured cells, biopsy or autopsy may be used. In the present invention, it can be used as a section to facilitate immunostaining. To increase the accuracy of immunostaining, the tissue antigen must be conserved. In one embodiment, if the antibody is detectable only in frozen tissue sections, the tissue can be frozen as soon as possible and then prepared for sections in frozen tissue.

In addition, tissues can be fixed and used for preservation of antigenicity. The type of fixing liquid used for fixing the tissue is not particularly limited. For example, a fixing liquid containing paraformaldehyde, glutaraldehyde, formalin, or methanol may be used. For the preservation of antigenicity, a suitable fixative is selected and the fixation time should be adjusted.

The type of the antibody that reacts with the antigen in the tissue is not particularly limited and can be appropriately selected depending on the type of tissue to be used. Generally, as an antibody, a monoclonal antibody or a polyclonal antibody can be used and can be used in a concentrated form or a diluted form. In the present invention, for example, Neun (neuronal nuclei) or MAB377 may be used as the above-mentioned antibody. In addition, the antibody is used in a diluted form. As the solvent for dilution, phosphate buffer (PBS) or sodium borate buffer may be used. The solvent may be Triton X-100 and bovine serum albumin (BSA) . The antibody in which the antibody is diluted may be referred to as an antibody solution. The antibody in the antibody solution may have a concentration of 1 to 10 占 퐂 / ml.

In the present invention, the antigen-antibody reaction is carried out under pressure. By performing the reaction under the above pressure, the antibody can penetrate deep into the tissue. At this time, the pressurization can be performed using positive pressure, negative pressure, centrifugal force, or a combination of these forces. The pressurization may be carried out through a separate pressurizing device, and the antigen-antibody reaction may be performed using a sealable kit to pressurize.

FIG. 1 is a schematic view illustrating a configuration of a kit according to an embodiment of the present invention. Referring to FIG.

As shown in FIG. 1, the kit may have a structure that can be sealed by a lid. There is a hydrogel bed inside the kit and tissues (antigens) can be placed on top of the hydrogel bed.

The hydrogel bed may consist of 4% acrylamide, 0.025% visacrylamide and 0.25% VA-0.44 initiator in PBS.

Acrylamide (acrylamide 40% solution, sigma, A4058), N, N-methylene-bis-acrylamide 2% solution and VA-044 initiator were used to prepare a final concentration of acrylamide 4%, bisacrylamide 0.025 %, VA-044 initiator 0.25%. In this case, phosphate buffer (PBS) is used as a solvent, but distilled water may be used instead of PBS. The hydrogel can be prepared by solidifying the prepared mixed solution at room temperature.

The thickness of the hydrogel bed may be 5 to 10 mm. In addition, the hydrogel bed may have a density similar to that of the tissue, and may specifically have an error range within ± 10%, ± 8%, or ± 5% of the density of the tissue. The movement of the tissue can be prevented through the hydrogel bed and the damage of the tissue can be prevented. In addition, since the hydrogel bed has a density similar to that of the tissue, permeation of the antibody solution is easy.

Membranes may be present on the side of the bed corresponding to the side where the tissue is located in the hydrogel bed, that is, the lower surface of the hydrogel bed.

In the kit, pressurization is performed, and the pressurization can be performed through a separate pressurizing device connected to the kit. As the pressurizing device, a centrifugal separator, a vacuum pump, or a filtration pump may be used. The pressurization may be positive pressure, negative pressure, centrifugal force, or a combination thereof as described above. In one embodiment, the pressurizing device may have a device configuration capable of repeatedly changing this force, if the pressurization is applied with a combined force of more than one of positive pressure, negative pressure and centrifugal force.

A pressure of 250 to 750 mmHg may be applied when the positive pressure is applied, a pressure of -500 to -250 mmHg when the negative pressure is applied, and a pressure of 600 to 3000 G when the pressure is applied by centrifugal force. A force of 10 to 50 Newtons (N) in terms of force units can be applied.

In one embodiment, a centrifuge can be used to pressurize with positive pressure, and a force of 600 to 3000 RCF can be applied to the kit.

The pressurization can be carried out for 1 to 8 hours, specifically for 1 to 2 hours.

In the tissue staining method of the present invention, a labeling agent for imaging may be incorporated into the antibody. As such a labeling agent, for example, an enzyme label, a fluorescent label, a radioisotope label or a gold colloid particle label can be used. Examples of the enzyme label include peroxidase, horseradish peroxidase or alkaline phosphatase. Examples of fluorescent labels include fluorescein isothiocyanate or fluorescein isothiocyanate. tetramethylrhodamine isothiocyanate can be used. Radioactive iodine such as ¹³¹ I and ¹²⁵ I may be used as the radioactive isotope.

The tissue staining method of the present invention can be further reacted with an antibody after performing the above-described antigen-antibody reaction. That is, an antigen in the tissue may be reacted with an antibody to bind the antibody with the antigen in the tissue, and then the antibody may be further reacted with the antibody bound to the antigen in the tissue.

In order to distinguish the two reactions, the reaction between the antigen and the antibody in the tissue can be referred to as a 'primary antigen-antibody reaction', and the reaction between the antibody and the antibody bound to the antigen in the tissue is referred to as a 'secondary antigen- can do. The antibody used in the first antigen-antibody reaction may be referred to as a primary antibody, and the antibody used in the secondary antigen-antibody reaction may be referred to as a secondary antibody.

In the present invention, the step of washing the tissue may be further performed before performing the first antigen-antibody reaction followed by the second antigen-antibody reaction. The washing may be performed to remove primary antibodies that have not reacted with the antigen in the tissue.

As the washing solution used for washing, for example, phosphate buffered saline (PBS) or the like can be used. The washing can be carried out using a washing method generally used in the art, for example, a method in which the above-described washing liquid is flowed after the tissue is separated from the kit can be used.

Further, in one embodiment, the washing may be performed by removing the primary antibody solution in the kit, adding the washing solution and pressing the washing solution. In this case, the washing can be carried out for 30 to 60 minutes, and the pressure can be increased to 600 to 3000 RCF when the centrifuge is used.

After the primary antigen-antibody reaction described above, a secondary antigen-antibody reaction is performed.

The secondary antibody used in the secondary antigen-antibody reaction may be an antibody against the immunoglobulin to which the primary antibody is attached. The type of the secondary antibody is not particularly limited, and fluorescence can be detected by emitting excitation light of a wavelength corresponding to each of the fluorescence isothiocyanate (FITC) and red fluorescence (Rhodamine). For example, it can be used ^{Aalexa Fluor ® 488 Donkey Anti-Mouse} IgG (H + L) Antibody or Cy3-AffiniPure F (ab ') 2 Frag Donkey Anti-Mouse IgG (H + L).

A labeling agent for imaging may be incorporated into the secondary antibody. The types of such markers are as described above.

The secondary antigen-antibody reaction can be performed using an antigen-antibody method that is generally performed in the art. Specifically, the secondary antibody solution diluted with the secondary antibody (the solvent mentioned above can be used as the solvent in the primary antibody solution) can be used to immerse the tissue to which the primary antibody is attached. In the secondary antibody solution, the secondary antibody may have a concentration of 1.5 to 3 μg / ml, and the secondary antigen-antibody reaction may be performed for 30 minutes to 48 hours, specifically 2 to 12 hours.

Also, in one embodiment, the secondary antigen-antibody reaction can be performed under pressure. The pressing conditions are as described above in the first antigen-antibody reaction. The secondary antigen-antibody reaction can be performed for 30 minutes to 5 hours, specifically 30 to 1 hour.

In addition, in the present invention, a step of washing the tissue after the second antigen-antibody reaction can be further performed.

As described above, the secondary antibody-antibody reaction is terminated by immersing the tissue in which the secondary antigen-antibody reaction has been completed in the washing solution, or the secondary antibody solution is removed from the kit in which the secondary antigen-antibody reaction is terminated under pressure. Washing can be carried out under pressure.

The tissues in which the above-described secondary antigen-antibody reaction has been completed are colored by the marker attached to the secondary antibody. The color of these tissues can be confirmed by a microscope or the like.

FIG. 2 and FIG. 3 are schematic views illustrating a procedure of performing a tissue dyeing method under a positive pressure condition (FIG. 2) or a negative pressure and positive pressure mixing condition (FIG. 3) according to an example of the present invention.

As shown in FIGS. 2 and 3, the primary antigen-antibody reaction (primary antibody reaction) and / or the secondary antigen-antibody reaction (secondary antibody reaction) are performed under positive pressure, Reaction can be carried out.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings, in which: FIG. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and is defined only by the scope of the claims of the invention.

Example 1

1 mm thick cerebral cortical sections were fixed in 4% paraformaldehyde (PFA) and then stained with phosphate buffered saline (PBS) containing 0.1% Triton X-100 for 3 times And washed.

Meanwhile, a primary antibody solution in which NeuN antibody was diluted to 1 ㎍ / ml was prepared. At this time, PBS containing 10% bovine serum albumin and 0.5% triton X-100 was used for dilution.

The washed cerebral cortical segment was placed on the hydrogel bed inside the kit. 400 μl of the primary antibody solution was dispensed into a kit, placed in a centrifuge (600 RCF), and allowed to react for 2 hours to perform a primary antigen-antibody reaction in the tissue section.

After removing the remaining primary antibody solution in the kit except for the hydrogel bed and the tissue section, the tissue was washed three times for 10 minutes using PBS.

A secondary antibody solution was prepared by diluting Aalexa Fluor 488 Donkey Anti-Mouse IgG (H + L) Antibody (A21202, Life technologies) to 2 μg / ml. PBS containing 10% bovine serum albumin and 0.5% triton X-100 was used for dilution.

After the secondary antibody solution was dispensed into the washed kit, the secondary antibody-antibody reaction was allowed to take place overnight at 4 ° C, and the tissue was washed with PBS.

Comparative Example 1

Tissue staining was performed in the same manner as in Example 1, except that the primary antigen-antibody reaction was performed for 2 hours without applying pressure.

Comparative Example 2

Tissue staining was performed in the same manner as in Example 1, except that the primary antigen-antibody reaction was performed for 2 days without applying pressure.

Example 2

(Alexa Fluor 488 donkey anti-rabbit IgG (H + L)) as the secondary antibody solution was diluted to 2 μg / ml with 1 μg / ml of microtubule-associated protein 2 Tissue staining was performed in the same manner as in Example 1, except that the diluted solution was used.

Example 3

Except that a solution of 1 μg / ml of CD31 was used as the primary antibody solution and a solution of Alexa Fluor 488 Donkey anti-Rat IgG (H + L) diluted to 2 μg / ml as the secondary antibody solution was used And tissue staining was carried out in the same manner as in Example 1.

4 is a photograph (A) of a tissue stained by the examples and comparative examples of the present invention, a graph (B) showing the intensity of fluorescence signal according to the degree of antibody penetration, and a graph showing the depth of tissue capable of quantitative analysis (C).

In FIG. 4, centrifugation (2-hr) is shown as an example, non-centrifugation (2 hr) as Comparative Example 1, and 2-day incubation as Comparative Example 2.

4A, it can be seen that the example in which the primary antigen-antibody reaction was performed for 2 hours by applying pressure to the tissue exhibited excellent color development as compared with the comparative example.

Also, FIG. 4B shows that the intensity of the immunostaining reaction signal is increased by 180% and the depth of tissue showing a significant signal intensity is increased by 210% compared to Comparative Example 1 in the case of the embodiment. This measurement shows that the rate of increase is 32% as compared with the case of Comparative Example 2 in which the antigen-antibody reaction is performed for 2 days.

That is, in order to obtain the dyeing effect through the usual experimental method, the time gain showed a time gain of 24 times or more.

Meanwhile, FIG. 4C shows that in the case of the embodiment, the depth of tissue capable of quantitative analysis is increased by 200% as compared with the case of Comparative Example 1, in which the immunochromatography at a level of 95% Can be confirmed. In addition, it can be confirmed that the rate of increase is 80% as compared with the case of Comparative Example 2. This means that the distribution of cells can be quantitatively analyzed and compared according to depth.

5 and 6, it can be seen that the dyeing method according to the present invention is applicable to various kinds of antibodies.

5A and 5B, it can be seen that the example in which the primary antigen-antibody reaction was performed for 2 hours by applying pressure to the tissue showed excellent color development as compared with the comparative example, It can be seen from C and D of FIG. 5 that the contrast is 240% deeper.

FIG. 6A shows that the result of the primary antibody-antibody reaction proceeded under pressure for 2 hours showed better color development than that of the comparative example. In the example, the depth of the stained tissue was 230% Deep can be confirmed through FIG. 6B.

4 through 6, it can be confirmed that the dyeing method of the present invention is applicable to various kinds of antibodies.

Example 4

In the same manner as in Example 1 except that negative pressure (negative pressure) and positive pressure were maintained at intervals of 5 minutes and repeatedly performed for 1 hour, Respectively.

Specifically, 1-mm-thick cerebral cortical sections were fixed in 4% paraformaldehyde (PFA), and then diluted in phosphate buffered saline (PBS) containing 0.1% Triton X-100 ≪ / RTI > three times.

Meanwhile, a primary antibody solution in which NeuN antibody was diluted to 1 ㎍ / ml was prepared. At this time, PBS containing 10% bovine serum albumin and 0.5% triton X-100 was used for dilution.

The washed cerebral cortical segment was placed on the hydrogel bed inside the kit. 1000 μl of the primary antibody solution was dispensed into a kit, and negative pressure and positive pressure were maintained at intervals of 5 minutes, and repeatedly applied pressure for 1 hour to induce primary antigen-antibody reaction in the tissue slice Respectively. At this time, a negative pressure was applied at a pressure of -500 to -250 mmHg, and a positive pressure was applied at a pressure of 250 to 750 mmHg.

After removing the remaining primary antibody solution in the kit except for the hydrogel bed and the tissue section, the tissue was washed three times for 10 minutes using PBS.

In addition, the ^{Aalexa Fluor ® 488 Donkey Anti-Mouse} IgG (H + L) Antibody (A21202, Life technologies) was prepared a secondary antibody solution diluted with 2㎍ / ㎖. PBS containing 10% bovine serum albumin and 0.5% triton X-100 was used for dilution.

After the secondary antibody solution was dispensed into the washed kit, the secondary antibody-antibody reaction was allowed to take place overnight at 4 ° C, and the tissue was washed with PBS.

Comparative Example 3

Tissue staining was performed in the same manner as in Example 1, except that the primary antigen-antibody reaction was performed for 1 hour without applying pressure.

7 is a photograph (A) of a tissue stained by Example 4 and Comparative Example 3 of the present invention, a graph (B) showing a fluorescence intensity of a fluorescent dye by antibody penetration, (C) showing the depth.

7, Negative-Positive pressure (1 hr) indicates Example 4, and Control (1 hr) indicates Comparative Example 3.

7, it can be seen that Example 4 in which the primary antigen-antibody reaction was performed for 1 hour by applying pressure to the tissue exhibited excellent color development as compared with Comparative Example 3.

7B, in Example 4, the intensity of the immunostaining signal was increased by 52% and the depth of tissue showing significant signal intensity was also increased by 212% compared with the comparative example. 7 shows that the dyed depth of the tissue is superior to the dyed depth of Comparative Example 3 in terms of the Z axis.

Claims (7)

In tissue staining,
A method of tissue staining comprising conducting the reaction of an antigen and an antibody in tissue under pressure.
The method according to claim 1,
Wherein pressurization is performed by pressurizing a kit in which the tissue is placed on top of the hydrogel bed.
3. The method of claim 2,
Wherein the hydrogel bed has a thickness of 5 to 10 mm.
3. The method of claim 2,
Wherein the density of the hydrogel bed comprises a tissue density and an error range of +/- 10%.
The method according to claim 1,
Wherein pressurization is carried out using positive pressure, negative pressure, centrifugal force or a combination of these forces.
6. The method of claim 5,
Wherein the positive pressure is from 250 to 750 mmHg, the negative pressure is from-500 to-250 mmHg, and the centrifugal force is from 600 to 3000 G force.
The method according to claim 1,
A method of tissue staining further comprising performing a reaction of an antibody in the tissue with an antigen and a secondary antibody under pressure.

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