TWI767587B - Peroxidase blocking buffer and peroxidase staining method by using ink-jet printing - Google Patents

Abstract

The disclosure provides a peroxidase blocking buffer. Its composition comprises, based upon weight percentage, a water soluble organic solvent of 70~90%, hydrogen peroxide of 0.5~10%, water of 0.4~20% and a surfactant of 0.1~0.5%, in which the blocking buffer has a viscosity range between 1 and 10 centipoise (cPs). The water soluble organic solvent is a primary material in the peroxidase blocking buffer of this disclosure and the peroxide blocking buffer has a better stability and blocking effect, compared to the conventional blocking buffer, so as to lower the background interference of picture after immunohistochemistry. Moreover, since the block buffer can be stably in ink cartridges, immunohistochemistry can be performed by automatic ink-jet printing.

Description

Peroxidase blocking agent and histochemical staining method using spray printing

The present invention relates to a peroxidase blocking agent, in particular to a blocking agent with better storage stability and a method for histochemical staining by spray printing.

Immunohistochemical staining technology (immunochemistry) is the application of the basic principle of immunology - antigen-antibody reaction, that is, the principle of specific binding of antigen and antibody. Its localization, qualitative or quantitative techniques. This histochemical staining technique is widely used in modern pathological diagnosis and plays a very important role.

Peroxidase is an enzyme commonly found in human organs and tissues, such as red blood cells, kidney and liver tissues. Currently, human tissue cells can be diagnosed by immunohistochemical staining (peroxidase stain; POX), such as diaminobenzidine (DAB) staining. The principle of diaminobenzidine staining method is to use peroxidase to oxidize hydrogen peroxide (hydrogen peroxide) to decompose it to release the original ecological oxygen (monatomic oxygen), and the original ecological oxygen will oxidize diaminobenzidine, Turn it into a brown-black granular precipitate, which can be used to diagnose and judge the activity of peroxidase through the shade of color. present In the conventional peroxidase dyeing method, water is used as the main substrate, for example, the content of water is about 95%, and after diluting and preparing the required hydrogen peroxide solution, the hydrogen peroxide solution is added to the hydrogen peroxide solution by the dropping method. Tissue sections were reacted with peroxidase. However, because the aqueous solution can promote the decomposition of hydrogen peroxide, the hydrogen peroxide solution prepared with water as the main matrix is not stable and is not easy to store, and tends to self-oxidatively decompose with the storage time, resulting in poor effect of blocking peroxidase. , which affects the high background value after histochemical staining and is not easy to interpret. Furthermore, since the hydrogen peroxide solution prepared with water as the main matrix decomposes by itself with the storage time, the results of histochemical staining before and after are also inconsistent. Because the hydrogen peroxide solution of this traditional blocking agent has an excessively high water content, the hydrogen peroxide solution easily reacts with the metal spring in the ink cartridge to generate gas, and the current peroxidase blocking agent cannot be filled in the ink cartridge. , hindering the possibility of using automated jet printing for histochemical staining.

Therefore, there is a need for a new and improved peroxidase blocking agent and a tissue staining method that can use a jet printing method to overcome the deficiencies in the prior art.

In view of this, an object of the present invention is to provide a peroxidase blocking agent, the composition of the blocking agent comprises in weight percentage: 70-99% water-soluble organic solvent, 0.5-10% hydrogen peroxide , 0.4-20% water, and 0.1-0.5% surfactant, wherein the blocking agent has a viscosity ranging from 1-10 centipoise (cPs). The peroxidase blocking agent of the embodiment of the present invention replaces the traditional blocking agent with water as the main matrix by a water-soluble organic solvent, thereby slowing down and reducing the decomposition of hydrogen peroxide, and improving the storage performance of the blocking agent. stability. That is to say, the peroxidase blocking agent of the embodiment of the present invention has a relatively high Excellent stability, the blocking effect is not affected by storage time, temperature, etc., and results with lower background values can be obtained after histochemical staining, which increases the accuracy of interpretation. In addition, because the peroxidase blocking agent of the embodiment of the present invention has better stability, the problem of different background values after tissue staining due to storage can be avoided, and the background value after staining can be maintained to be consistent.

Preferably, the water-soluble organic solvent of the peroxidase blocking agent comprises alcohols, alcohol ethers or diethers.

Preferably, the alcoholic water-soluble organic solvent of the peroxidase blocking agent comprises monoalcohols or polyalcohols.

Preferably, the water-soluble organic solvent of the alcohols of the peroxidase blocking agent is selected from methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, diethylene glycol, triethylene glycol Alcohol, Tetraethylene Glycol, Polyethylene Glycol, 1,2-Propanediol, 1,3-Propanediol, Dipropylene Glycol, Tripropylene Glycol, Polypropylene Glycol, Glycerol, 1,2-Butanediol, 1,3-Butanediol Diol, 1,4-Butanediol, 2,3-Butanediol, Butanetriol, Neopentyl Glycol, 1,5-Pentanediol, Pentapentanol, 1,6-Hexanediol and Hexanehexanol group of alcohols.

Preferably, the water-soluble organic solvent of the alcohol ethers of the peroxidase blocking agent comprises a unilateral end cap with methyl, ethyl, n-propyl, isopropyl, n-butyl or trimethyl. tertiary butyl polyol.

Preferably, the water-soluble organic solvent of the alcohol ethers of the peroxidase blocking agent is selected from ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monopropyl ether, and ethylene glycol monobutyl ether. ether, ethylene glycol mono-tert-butyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monobutyl ether, polyethylene glycol monomethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, The group consisting of dipropylene glycol monomethyl ether, dipropylene glycol monopropyl ether, dipropylene glycol monobutyl ether and tripropylene glycol monomethyl ether.

Preferably, the water-soluble organic solvent of the diethers of the peroxidase blocking agent comprises polyhydric alcohols at the end caps of at least two sides.

Preferably, the water-soluble organic solvent of the diethers of the peroxidase blocking agent is selected from ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol The group consisting of diethyl ether, diethylene glycol methyl ethyl ether, triethylene glycol dimethyl ether, tetraethylene glycol dimethyl ether and dipropylene glycol dimethyl ether.

Preferably, the surfactant of the peroxidase blocker comprises polysorbate (Tween 20).

Furthermore, the peroxidase blocking agent of the embodiment of the present invention uses a water-soluble organic solvent as the matrix of the blocking agent, which reduces the possibility of reacting with the ink cartridge, such as a metal spring, to generate gas, so it can be loaded more stably. Filled in the ink cartridge, it is possible to realize the histochemical staining method using the automatic spray printing method. In addition, filling the peroxidase blocking agent in the ink cartridge can effectively isolate the blocking agent from contact with the outside air, and at the same time, it can also improve the storage stability of the peroxidase blocking agent.

Another object of the present invention is to provide a histochemical staining method using a jet printing method, the steps of which include: providing a carrier; placing a tissue sample on the carrier; detecting the tissue sample to define a jet printing area; printing a peroxidase blocking agent to the printing area; and printing a dyeing agent to the printing area for histochemical staining; wherein, the composition of the peroxidase blocking agent is by weight The percentage meter includes: 70~99% water-soluble organic solvent, 0.5~10% hydrogen peroxide, 0.4~20% water, and 0.1~0.5% surfactant, and the peroxidase blocking agent has A viscosity range from 1 to 10 centipoise (cPs). The histochemical staining is carried out by the spray printing method of the automatic machine, and the blocking agent can be accurately printed in the area to be printed, which can greatly reduce the amount of blocking agent, avoid the waste of blocking agent, and save costs.

Preferably, in the histochemical staining method using jet printing, the tissue sample comprises a biological tissue sample containing peroxidase.

Preferably, in the histochemical staining method using a jet printing method, the detecting step includes photographing the tissue sample through a lens, and defining the jet printing area through identification.

Preferably, in the histochemical staining method using jet printing, the staining agent comprises diaminobenzidine (DAB).

Preferably, in the histochemical staining method using jet printing, the step of jet printing the dye is after jet printing the peroxidase blocking agent.

2: Bearing seat

4: Tissue samples

6: Detection lens

8: Inkjet head

10: Blockers

12: Dye

14: Tissue samples

1A-1B are schematic photographs of histochemical staining using a traditional blocking agent and a peroxidase blocking agent according to an embodiment of the present invention.

Figure 2 is a schematic diagram of the blocking effect of the peroxidase blocking agent of the example of the present invention.

3A-3D are schematic diagrams illustrating steps of performing histochemical staining by spray printing according to another embodiment of the present invention.

In order to understand the technical features and practical effects of the present invention in detail, and to implement in accordance with the contents of the description, further preferred embodiments are described in detail as follows with the drawings. It should be understood that the numerical values or ranges in the following embodiments are only used to illustrate the examples in which the present invention can be implemented, and those skilled in the art The values or ranges of the embodiments may be slightly adjusted and changed as required, and therefore, the contents of the embodiments are not intended to limit the present invention.

The embodiment of the present invention discloses a peroxidase blocking buffer with better stability. First, take the water-soluble organic solvent of 70~90% by weight as the main matrix component of the blocking agent, then add the aqueous hydrogen peroxide diluted with water, wherein the weight percent of water is 0.4~20%, and the hydrogen peroxide is 0.4~20% by weight. The weight percentage is 0.5~10%. Finally, 0.1-0.5% by weight of surfactant is added to the above solution, and stirred for about 5 minutes to prepare the peroxidase blocking agent according to the embodiment of the present invention. The matrix here refers to the most important component in the composition blocking agent, and it can also refer to, for example, that the composition weight percentage of the composition is more than 50%. In another embodiment, the weight percentage of the surfactant can also be adjusted to 0.01-2%, preferably 0.05%, and the weight percentage of the above-mentioned hydrogen peroxide can also be preferably 3-5%.

The above-mentioned water-soluble organic solvent may preferably be alcohols, alcohol ethers or diethers. In the embodiment where the alcohols are water-soluble organic solvents, they can be monoalcohols or polyhydric alcohols, and preferably can be selected from methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, diethyl alcohol Glycol, Triethylene Glycol, Tetraethylene Glycol, Polyethylene Glycol, 1,2-Propanediol, 1,3-Propanediol, Dipropylene Glycol, Tripropylene Glycol, Polypropylene Glycol, Glycerol, 1,2-Butanediol , 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, butanetriol, neopentyl glycol, 1,5-pentanediol, pentanepentanol, 1,6- The group consisting of hexylene glycol and hexanol. However, in the embodiment in which the alcohol ether is a water-soluble organic solvent, it can be a polyol with methyl, ethyl, n-propyl, isopropyl, n-butyl or tertiary butyl at the end capped at one end. , and preferably can be selected from ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monopropyl ether, ethylene glycol monobutyl ether, ethylene glycol mono-tert-butyl ether, diethylene glycol monomethyl ether , Diethylene glycol monoethyl ether, diethylene glycol monobutyl ether, polyethylene glycol monomethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, dipropylene glycol mono The group consisting of methyl ether, dipropylene glycol monopropyl ether, dipropylene glycol monobutyl ether and tripropylene glycol monomethyl ether. In another embodiment in which diethers are used as water-soluble organic solvents, it can be diethers containing polyhydric alcohols at least on both sides of the end caps, and preferably can be selected from ethylene glycol dimethyl ether, ethylene glycol Group consisting of diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, diethylene glycol methyl ethyl ether, triethylene glycol dimethyl ether, tetraethylene glycol dimethyl ether and dipropylene glycol dimethyl ether Group. It can be understood that the above examples of the water-soluble organic solvent are only used to illustrate the specific implementation mode of the present invention, and other organic solvents with similar properties are also possible, and are not intended to limit the present invention. The above-mentioned surfactant can preferably be polysorbate of Tween 20 or other suitable nonionic surfactant.

After the peroxidase blocking agent of the embodiment was prepared, a tissue sample was taken for immunohistochemistry (IHC). The peroxidase blocking agent of the above-mentioned embodiment is added to the tissue sample section, and after about 15 to 30 minutes, the peroxidase of the tissue sample section is allowed to react with the blocking agent, and then the dye is added to the above-mentioned tissue. Slice the sample and let it stand for about 5 to 10 minutes. After the reaction has developed, the staining status of the tissue sample can be observed. In one embodiment, the above-mentioned dye may preferably be diaminobenzidine (DAB) or other similar dyes. However, the tissue sample may be any tissue sample containing peroxidase, such as liver or kidney tissue, blood cells, and the like. It is worth mentioning that, before adding the dye, the peroxidase blocking agent can be washed and removed with a detergent. The above-mentioned detergent may preferably be phosphate buffered saline (PBST) or Tris-HCl buffered saline (TBST).

Figures 1A-1B are schematic diagrams of photographs after histochemical staining after blocking with a conventional peroxidase blocking agent and a peroxidase blocking agent according to an embodiment of the present invention, respectively. FIG. 1A is a control group using a conventional peroxidase blocking agent, and FIG. 1B is a photo of histochemical staining using the peroxidase blocking agent of this example. As can be seen from Figure 1A, the group using the conventional peroxidase blocker The background value of tissue staining is high, and its blocking effect is poor, while the peroxidase blocking agent of the embodiment of the present invention has a better blocking effect, so that the background value of the tissue sample after staining is low, as shown in Figure 1B . The blocking agent of the embodiment of the present invention can improve and avoid problems such as affecting the interpretation of staining results due to high background value. Furthermore, the peroxidase blocking agent of this embodiment has better stability, and does not cause inconsistency in histochemical staining results before and after the blocking agent is decomposed due to storage time. Detergent does make histochemical staining results more consistent with background values.

Next, the present invention provides a plurality of experimental examples of blocking agents with different formulations, and observes the blocking effect (background blocking) of peroxidase blockers under different formulations. Table 1 shows a total of 10 groups of experimental examples. Except for the Tween 20 weight of the surfactant, which remains unchanged, other components such as water-soluble organic solvents, water, and hydrogen peroxide are all different in these 10 groups of experimental examples. According to the above-mentioned steps, the peroxidase blocker of different formulations is prepared, such as Experimental Example 1 or Example 1, take 98.45 grams of water-soluble organic solvent of diethylene glycol diethyl ether, and add 0.92 grams of water and 0.5 grams of water. Hydrogen peroxide, and 0.13 grams of Tween 20, were mixed and stirred for about 5 minutes to obtain the peroxidase blocker of Example 1. Then, according to the above steps, the peroxidase blocking agent of Example 1 was added to the tissue section, and after about 15-30 minutes of reaction, the dyeing agent of diaminobenzidine (DAB) was added, and it was allowed to stand for about 5-30 minutes. After 10 minutes of staining, the background value and blocking effect of the tissue staining results can be observed. Other examples 2 to 10 can be prepared according to this procedure and histochemically stained to observe the background value and blocking effect. It is worth mentioning that, it can also be a water-soluble organic solvent as a blocking agent that replaces the diethylene glycol diethyl ether in each example of Table 1 with any organic solvent in the above-mentioned alcohols, alcohol ethers and diethers. . Therefore, the water-soluble organic solvent of the present invention is also not limited to the disclosed components of the examples in Table 1.

Table 1

In Figure 2, it is a schematic diagram of the blocking effect of Examples 1-10 in Table 1, and the two groups are respectively the control group without adding any blocking agent and the histochemically stained control group with the addition of a conventional blocking agent. Here, the conventional peroxidase blocking agent is a blocking agent with an aqueous solution as the main matrix, so the weight percentage of water is relatively high, so that the conventional blocking agent is easier to decompose, has poor stability and is not easy to store, The resulting poor blocking effect is shown in Figure 2. In the figure, the blocking agent of the examples 1 to 10 proposed by the present invention adopts the water-soluble organic solvent of diethylene glycol diethyl ether as the matrix component of the peroxidase blocking agent, all of which are better than the control group, without adding Blocking effects of blockers and conventional blockers. In particular, the blocker formulation composition of Example 4 has more than 50% blocking effect. Thus, it can be seen that the peroxidase blocking agent using water-soluble organic solvent instead of the conventional blocking agent and using water as the main matrix of the embodiment of the present invention indeed has a significantly better blocking effect than the conventional blocking agent. Here, the blocking effect can refer to the background value after histochemical staining. A low background value can be inferred that the peroxidase blocking agent has a better blocking effect. On the contrary, a high background value can be inferred that the blocking effect of the blocking agent. The interruption effect is poor.

Since the peroxidase blocking agent in the embodiment of the present invention uses a water-soluble organic solvent as the main matrix component, it can reduce or avoid the chance of generating gas by reacting with, for example, a metal spring in the ink cartridge, and can be stably filled and loaded in the ink cartridge. In the ink cartridge, the automatic inkjet method for histochemical staining method can be realized. In order to make the peroxidase blocking agent of the embodiment of the present invention suitable for spraying by inkjet, it can have a viscosity range of 1-10 centipoise (cPs), and a preferable viscosity range is 1-5 centipoise (cPs). It can be understood that, as long as the inkjet system is not affected, the viscosity of the peroxidase blocking agent can also be in other suitable ranges, and is not limited to the above range. Next, an embodiment of the present invention provides a histochemical staining method for printing the above-mentioned peroxidase blocking agent using an inkjet method.

3A-3D are schematic flowcharts of a histochemical staining method using an inkjet method according to another embodiment of the present invention. As shown in FIG. 3A , a carrier 2 such as a glass slide is provided, and a tissue sample 4 is placed on the carrier 2 , and then the tissue sample 4 is detected to define a printing area. In one embodiment, a photo of the tissue sample 4 can be taken through, for example, the detection lens 6, and the position to be printed can be identified through a computer or the detection lens can be directly scanned to detect and define the spray of the tissue sample 4. print area. The method of detecting the tissue sample is not particularly limited here, as long as the printing area is obtained and defined in an appropriate manner. After the print area is defined, the peroxidase blocking agent 10 is sprayed onto the print area of the tissue sample 4 through an inkjet head 8, as shown in FIG. 3B . The blocking agent 10 can react with the peroxidase in the tissue sample 4 to block the reaction. In one embodiment, the peroxidase blocking agent may have a viscosity in the range of 1 to 10 centipoise (cPs), and a preferred viscosity range may also be in the range of 1 to 5 centipoise (cPs). cPs). Afterwards, as shown in FIG. 3C , through the inkjet head 8 , the dye 12 is sprayed onto the print area of the tissue sample 4 to perform histochemical staining. In FIG. 3D , the tissue sample 14 after histochemical staining by ink jet is shown. In this embodiment, the dyeing agent 10 and the tissue sample 4 can be made of the same or similar materials as those in the above-mentioned embodiment, which will not be repeated here.

Since the embodiment of the present invention uses inkjet printing, such as peroxidase blocking agent, etc., the blocking agent can be accurately printed. For example, by controlling the position to be printed and the amount of blocking agent, the blocking agent can be greatly reduced. Amount of breaker used. Furthermore, in this embodiment, the peroxidase blocking agent can be filled in a high-sealed ink cartridge to reduce the chance of the blocking agent being decomposed due to contact with air, so that the peroxidase blocking agent can also be improved. Storage stability. Besides the blocking agent can be spray-printed, in the embodiment of the present invention, the dyeing agent can also be added to the tissue sample by automatic spray-printing method, so the amount of the dyeing agent can be saved at the same time, so the overall reduction of histochemical staining can be achieved. method cost.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the scope of rights claimed by the present invention. All other equivalent changes or modifications that do not depart from the spirit disclosed in the present invention shall be included in the present invention. within the scope of the patent application.

Claims (13)

A peroxidase blocking agent, its composition comprises by weight percentage: 70-99% of a water-soluble organic solvent, 0.5-10% of hydrogen peroxide, 0.4-20% of water, and 0.1-0.5% of water A surfactant, wherein the blocking agent has a viscosity ranging from 1 to 10 centipoise (cPs). The peroxidase blocking agent according to claim 1, wherein the water-soluble organic solvent comprises alcohols, alcohol ethers or diethers. The peroxidase blocking agent of claim 2, wherein the water-soluble organic solvent of alcohols is selected from methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, diethylene glycol, Ethylene Glycol, Tetraethylene Glycol, Polyethylene Glycol, 1,2-Propanediol, 1,3-Propanediol, Dipropylene Glycol, Tripropylene Glycol, Polypropylene Glycol, Glycerol, 1,2-Butanediol, 1,3 -Butanediol, 1,4-butanediol, 2,3-butanediol, butanetriol, neopentyl glycol, 1,5-pentanediol, pentanepentanol, 1,6-hexanediol and A group consisting of hexanol. The peroxidase blocking agent as claimed in claim 2, wherein the water-soluble organic solvent of alcohol ethers comprises a single-side end cap with methyl, ethyl, n-propyl, isopropyl, n-butyl Or tertiary butyl polyol. The peroxidase blocking agent of claim 4, wherein the water-soluble organic solvent of alcohol ethers is selected from ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monopropyl ether, ethylene glycol Monobutyl ether, ethylene glycol mono-tert-butyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monobutyl ether, polyethylene glycol monomethyl ether, propylene glycol monomethyl ether, propylene glycol mono The group consisting of diethyl ether, dipropylene glycol monomethyl ether, dipropylene glycol monopropyl ether, dipropylene glycol monobutyl ether and tripropylene glycol monomethyl ether. The peroxidase blocking agent according to claim 2, wherein the water-soluble organic solvent such as diethers comprises polyhydric alcohols at the end caps on at least two sides. The peroxidase blocking agent according to claim 6, wherein the water-soluble organic solvent of diethers is selected from ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethyl ether The group consisting of glycol diethyl ether, diethylene glycol methyl ethyl ether, triethylene glycol dimethyl ether, tetraethylene glycol dimethyl ether and dipropylene glycol dimethyl ether. The peroxidase blocking agent of claim 1, wherein the surfactant comprises polysorbate (Tween 20). A histochemical staining method using jet printing, the steps of which include: providing a bearing seat; placing a tissue sample on the bearing seat; detecting the tissue sample to define a jet printing area; jet printing as requested The peroxidase blocking agent of 1 is sprayed on the printing area; and a dye is sprayed on the printing area for histochemical staining. The histochemical staining method using jet printing according to claim 9, wherein the tissue sample comprises a biological tissue sample containing peroxidase. The histochemical staining method using a jet printing method according to claim 9, wherein the detecting step comprises photographing the tissue sample through a lens, and defining the jet printing area through identification. The histochemical staining method using jet printing according to claim 9, wherein the staining agent comprises diaminobenzidine (DAB). The histochemical staining method using jet printing according to claim 9, wherein the step of jet printing the dye is after jet printing the peroxidase blocking agent.

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