CN101556224B - Manufacturing method of paraffin tissue chip - Google Patents

Abstract

The invention provides a method for making a paraffin tissue chip, which is characterized in that the specific steps are: numbering the required tissues and then immersing them in wax, or marking the required tissues on the existing wax blocks and then numbering them; cutting the tissues into pieces Put the desired size on the ironing table, and melt the paraffin on it at 60-65°C to obtain the tissue to be embedded; take another paraffin and put it into a paraffin pot, heat it with an electric furnace to melt the paraffin; put the square wax mold on the glass On the board, arrange the tissues to be embedded obtained in the first step in the melted paraffin and arrange them on one side of the wax mold, and record the number of the tissue at the corresponding position on the positioning board or positioning paper. Leave a square space on the other side, pour melted paraffin into the wax mold along the space, until the tissue is submerged to polymerize the paraffin, remove the wax mold, and slice along the direction parallel to the glass plate. The invention has the advantages of low cost, convenience, speed, high output and various materials, and can be popularized in every pathology department and related research units.

Description

A kind of preparation method of paraffin tissue chip

technical field

The invention relates to a method for preparing a paraffin tissue chip, which is used for preparing a tissue chip and belongs to the technical field of tissue chips.

Background technique

Tissue chip technology is the development and extension of gene chip technology. Like cell chip, protein chip and antibody chip, it belongs to a special biochip technology. Tissue chip technology can arrange dozens or even thousands of clinical tissue specimens of different individuals in a pre-designed sequence for analysis and research. It is a high-throughput, multi-sample analysis tool. Tissue chip technology enables researchers to analyze hundreds or even thousands of tissue samples at the same time, and conduct research on one or more specific genes or their related expression products. This technology can be combined with DNA, RNA, protein, antibody and other technologies, combined with traditional pathology technology, histochemistry and immunohistochemistry technology, and can be carried out at the three levels of gene, gene transcription and related expression product biological functions. Research. This has practical significance for the research and development of human genomics, especially the study of the relationship between genes and proteins and diseases, the verification of disease-related genes, the development and screening of new drugs, the molecular diagnosis of diseases, the tracking and prognosis of treatment processes, etc. and broad market prospects.

At present, a few biotechnology companies such as Clontech and Stratagene in the United States have developed and sold human and animal tissue chip products, but the quantity is small, the price is high, and the variety is single, which cannot meet the needs of medical research and pharmaceutical industry research and development. In April 2001, American LifeSpan Biosciences established a database of gene expression in normal and diseased tissues. American Tissuelnformactics company uses animal tissue chip technology to screen drug toxicity and find new drug action sites. In addition, Japan, the United Kingdom and other countries are actively preparing to build national clinical histopathology databases.

China's research on tissue chip technology is progressing rapidly. In October 2001, the Ministry of Science and Technology of China listed tissue chip technology as a major project for the development of the western part of the national scientific and technological research in the "Tenth Five-Year Plan", and the project has been officially established in Xi'an. The project is divided into 7 sub-projects, including tissue chip technology, tissue chip biological information database, automated tissue chip instrument, automatic analysis device, tissue chip related technology and reagents, tissue chip practical application technology, and laid a solid foundation for the simultaneous development of related technologies in China and the world. Base.

At present, the preparation of tissue chips mainly relies on mechanized chip preparation instruments. The preparation instrument includes an operating platform, a special punching sampling device and a positioning system. The punching sampling device samples the donor tissue wax block, and can also punch the recipient wax block at the same time. The hole diameter is the same as the sampling diameter, and both can be precisely positioned. The positioning device of the preparation instrument can move the puncture needle or the acceptor wax block linearly, so as to prepare tissue microarray wax blocks with the same pore diameter, hole distance and hole depth. It is transferred and fixed on siliconized and gelled glass slides through the slice auxiliary system to become a tissue chip. According to different sample diameters (0.2-2.0mm), more than 40-2000 tissue samples can be arranged on a 45mm×25mm glass slide. Generally, tissue microarrays are divided into low-density microarrays (<200 points), medium-density microarrays (200-600 microarrays) and high-density microarrays (>600 microarrays) according to the number of samples. Commonly used tissue microarrays contain between 50 and 800 tissue samples. According to different research purposes, the types of chips can be divided into dozens of tissue chips such as tumor tissue chips, normal tissue chips, single or composite, and specific pathological types.

However, the current preparation of tissue chips according to the above method has the following disadvantages: first, the production cost of existing tissue chips is very high. In addition to special equipment (hundreds of thousands of yuan), the cost of making a tissue chip is more than 10,000 yuan, and the unit price of each slice is 50-100 yuan; The source is limited; thirdly, the chip prepared according to the existing equipment and method has only about 50 slices, because the sample is made by punching holes on the original wax block, and the tissue thickness is limited; fourthly, although the high-density chip has a large throughput, However, due to the small size of each tissue, it is difficult to provide information on the tissue structure; fifth, only large-scale research units have the funds and conditions to conduct related research on tissue chips, which is difficult to promote.

Contents of the invention

The purpose of the present invention is to develop a simple tissue chip manufacturing method to prepare low-density and medium-density chips, taking into account the requirements of sample throughput, tissue structure and chip output, so that the role of tissue chip technology can be fully exerted.

In order to achieve the above object, the technical solution of the present invention is to provide a method for making a paraffin tissue chip, characterized in that, the specific steps are:

Step 1: Dip the required tissue in paraffin after numbering, or mark the required tissue on the existing wax block and then number; cut the tissue into the required size, put it on the ironing table, and heat it at 60-65°C The paraffin on it is melted to obtain the tissue to be embedded;

Step 2: Take another paraffin and put it into a paraffin pot, and heat it to 60-70°C with an electric furnace to melt the paraffin;

Step 3: Place the square wax mold on the glass plate, immerse the tissue to be embedded obtained in the first step in the paraffin wax melted in the second step, and then arrange it on one side of the wax mold, place it on the positioning plate or Record the number of the tissue at the corresponding position on the positioning paper, leave a square space on the other side of the wax mold, and pour the melted paraffin into the wax mold along the space until the tissue is submerged. ) to stand for 10-30min to allow the paraffin to polymerize, remove the wax model, and slice along the direction parallel to the glass plate.

Further, the tissue cut in the first step is in the shape of a cube or a cylinder.

The cube shape has a length of 5 mm to 10 mm, a width of 1 mm to 2 mm, and a thickness of 1 mm to 2 mm.

The wax mold in the third step is composed of two vertically bent copper strips, the length is 70mm, the width is 40mm, the height is 20mm, and the thickness is 10-20mm.

The length of the square space left in the third step is 20 mm, and the width is 25 mm.

The present invention makes use of the characteristics of easy dissolution and polymerization of paraffin to arrange the required tissues in a certain order. First, a small amount of paraffin is used to loosely adhere the tissues together, and finally the tissues are completely polymerized with paraffin, and the required tissues are obtained after sectioning. chip.

The invention has the advantages of low cost, convenience, speed, high output and various materials, and can be popularized in every pathology department and related research units. The tissue chip produced is unlimited in shape and can be long, rectangular, square, cylindrical, cylindrical or irregular; the size of the tissue can be determined according to the needs, and 20 cells can be arranged on an area of 50mm×25mm. ~300 organizations can meet research needs. The height of the tissue chip can also be determined according to the needs, and 500 to 2000 slices can be cut on a tissue chip with a height of 5 mm to 20 mm, which is much higher than the output of 50 slices cut by the existing tissue chip, which is about 10 to 40 times; the cost is much lower than the existing production methods.

Detailed ways

The present invention will be described in detail below in conjunction with the examples.

Example 1

Paraffin tissue chips were prepared by the following method:

Step 1: Take the required tissues when collecting pathological materials, number them, cut them into a size of 2mm×2mm×10mm, follow the routine tissue processing procedures of the pathology department, and go to the step of soaking them in paraffin; put them on the ironing table at 60°C The paraffin on the surface is melted to obtain the tissue to be embedded;

Step 2: Take another paraffin and put it into a paraffin pot, and heat it to 60°C with an electric furnace to melt the paraffin;

Step 3: Put 2 vertically bent copper strips on a thick glass plate, put together a square wax mold with a length of 70mm, a width of 40mm, a height of 20mm, and a thickness of 10mm on a glass plate, with a thickness of (copper The height of the strip), use tweezers to take the first tissue to be embedded, quickly immerse it once in melted paraffin, stick the flat surface to the glass, rely on the stickiness of the paraffin to stand the tissue on the glass, the first tissue has The two sides are close to the wax model, the bottom is close to the glass, and the original number of the tissue is recorded on the positioning board or positioning paper; the second tissue is placed in turn, the bottom of the second tissue is close to the glass, and one side is the same as the first The two tissues are connected, and one side is connected with the wax model. The distance between the two tissues is equivalent to the distance between the two layers of wax models, until the first row of tissue points is completed, and the length is 50mm. Then start the second row, the third row, etc., until the width of the wax pattern reaches 25mm, and there is a space of 20mmx25mm in the wax pattern. Slowly pour the paraffin melted in the paraffin pot into the wax pattern along the space until it is completely submerged; Stand at 25°C for 10 minutes to polymerize the paraffin, remove the wax model, and prepare the wax block embedding n tissues, and slice along the direction parallel to the glass plate.

Example 2

Paraffin tissue chips were prepared by the following methods:

Step 1: Number and position the required tissue on the wax block, cut the tissue into a square block with a length of 5mm, a width of 1mm, and a thickness of 1mm; put it on the ironing table, and melt the paraffin on it at 65°C Obtain the tissue to be embedded;

Step 2: Take another paraffin and put it into a paraffin pot, and heat it to 70°C with an electric furnace to melt the paraffin;

Step 3: Put 2 vertically bent copper strips on a thick glass plate, put together a square wax mold with a length of 70mm, a width of 40mm, a height of 20mm, and a thickness of 20mm on a glass plate, with a thickness of (copper The height of the strip), use tweezers to take the first tissue to be embedded, quickly immerse it once in melted paraffin, stick the flat surface to the glass, rely on the stickiness of the paraffin to stand the tissue on the glass, the first tissue has The two sides are close to the wax model, the bottom is close to the glass, and the original number of the tissue is recorded on the positioning board or positioning paper; the second tissue is placed in turn, the bottom of the second tissue is close to the glass, and one side is the same as the first The two tissues are connected, and one side is connected to the wax model. The distance between the two tissues is equivalent to the distance between the two layers of wax models, until the first row of tissue points is completed, and the length is 50mm. Then start the second row, the third row, etc. until the width of the wax pattern reaches 25mm, and there is a space of 20mmx25mm in the wax pattern. Slowly pour the paraffin melted in the paraffin pot into the wax pattern along the space until it is completely submerged; Stand at 25°C for 10 minutes to polymerize the paraffin, remove the wax model, and prepare the wax block embedding n tissues, and slice along the direction parallel to the glass plate.

Example 3

Paraffin tissue chips were prepared by the following method:

Step 1: Number and position the required tissue on the wax block, cut the tissue into a cylinder with a length of 10mm and a radius of 1mm; put it on the ironing table, and melt the paraffin on it at 65°C to obtain the tissue to be embedded organization;

Step 2: Take another paraffin and put it into a paraffin pot, and heat it to 70°C with an electric furnace to melt the paraffin;

Step 3: Put 2 vertically bent copper strips on a thick glass plate, put together a square wax mold with a length of 70mm, a width of 40mm, a height of 20mm, and a thickness of 20mm on a glass plate, with a thickness of (copper The height of the strip), use tweezers to take the first piece of tissue to be embedded, quickly immerse it once in melted paraffin, stick the flat surface to the glass, rely on the stickiness of the paraffin to stand the tissue on the glass, the first piece of tissue has two The side is close to the wax model, the bottom is close to the glass, and the original number of the tissue is recorded on the positioning board or positioning paper; the second tissue is placed in turn, the bottom of the second tissue is close to the glass, and one side is connected to the first tissue , the other side is connected to the wax model, the distance between the two tissues is equivalent to the distance between the two layers of wax models, until the first row of tissue points is completed, and the length is 50mm. Then start the second row, the third row, etc. until the width of the wax pattern reaches 25mm, and there is a space of 20mmx25mm in the wax pattern. Slowly pour the paraffin melted in the paraffin pot into the wax pattern along the space until it is completely submerged; After standing at 18°C for 30 minutes to allow the paraffin to polymerize, the wax model was removed, and the wax blocks embedding n tissues were prepared, and sliced along the direction parallel to the glass plate.

Claims (5)

1. a manufacturing method of paraffin tissue chip is characterized in that, concrete steps are:

The first step:, or numbering after the mark tissues needed on the existing wax stone with tissues needed numbering back paraffin; Tissue is cut into required size, is placed on and scalds on the platform, melt the tissue that obtains treating embedding at the 60-65 ℃ of paraffin on it;

Second step: other gets paraffin and puts into the paraffin kettle, is heated to 60-70 ℃ with electric furnace paraffin is melted;

The 3rd step: square wax-pattern is placed on the glass plate, the side that is arranged in wax-pattern inside in the paraffin that melts in second step after the submergence that is organized in for the treatment of embedding that the first step is obtained, the numbering of the tissue of record relevant position on location-plate or location paper, other side in wax-pattern inside stays square room, the paraffin that melts is poured in the wax-pattern along empty place, until organizing submergence, leaves standstill 10-30min at 18-25 ℃ and makes the paraffin polymerization, remove wax-pattern, along being parallel to the section of glass plate direction.

2. a kind of manufacturing method of paraffin tissue chip as claimed in claim 1 is characterized in that, is organized as Fang Tizhuan or cylindrical shape after cutting in the described first step.

3. a kind of manufacturing method of paraffin tissue chip as claimed in claim 2 is characterized in that, the length of described Fang Tizhuan is 5mm～10mm, the wide 1mm～2mm of being, the thick 1mm～2mm of being.

4. a kind of manufacturing method of paraffin tissue chip as claimed in claim 1 is characterized in that, the wax-pattern in described the 3rd step is made up of the copper bar of 2 vertical bendings, and long is 70mm, and wide is 40mm, and height is 20mm, and thick is 10～20mm.

5. a kind of manufacturing method of paraffin tissue chip as claimed in claim 2 is characterized in that, the length in the square room that stays in described the 3rd step is 20mm, and wide is 25mm.