CN215628008U - Paraffin-embedded tissue sample cell nucleus extraction kit - Google Patents

Abstract

The utility model discloses a paraffin-embedded tissue sample cell nucleus extraction kit which comprises a kit body, a proteolytic enzyme reagent bottle, a buffer solution reagent bottle, an eluent A reagent bottle, an eluent B reagent bottle, an eluent C reagent bottle, an eluent D reagent bottle, a fixed solution reagent bottle and a fixing part with seven reagent bottle clamping grooves, wherein the fixing part is detachably or non-detachably arranged in the kit body, the seven reagent bottles are respectively fixed by the seven reagent bottle clamping grooves, the eluent A is dimethylbenzene, the eluent B is absolute ethyl alcohol or an ethyl alcohol solution with the concentration of more than 85%, the eluent C is an ethyl alcohol solution with the concentration of 65% -84%, the eluent D is distilled water, and the fixing solution is formalin. The kit or the kit of reagent consumables can be used together with a cell nucleus staining reagent for DNA ploidy analysis and/or chromatin configuration analysis.

Description

Paraffin-embedded tissue sample cell nucleus extraction kit

Technical Field

The utility model relates to a cell nucleus extraction reagent, in particular to a cell nucleus extraction kit for a paraffin-embedded tissue sample.

Background

Paraffin-embedded tissue samples are the most common tumor pathological tissue samples at present, and in order to diagnose the tumor properties, pathologists usually extract DNA or RNA from the paraffin-embedded tissue samples for molecular biological analysis, such as single nucleotide polymorphism analysis, DNA ploidy analysis, RNA expression analysis, and the like. The existing research shows that the DNA ploidy is related to the prognosis of a patient, the existing DNA ploidy analysis method is mainly carried out by flow cytometry, but the DNA ploidy quantification obtained by flow cytometry detection is only a quantitative result, cannot explain the dynamic cause, cannot further explain the same fold risk degree, and may be different. In fact, disordered arrangement, changed direction, thickening, cracking and the like of nuclear chromatin are all the reasons for the change of DNA ploidy and have little influence on the prognosis of tumors. Therefore, when tumor prognosis is evaluated by DNA changes in tumor cells, it is not enough to focus on DNA ploidy alone, and DNA morphological structure changes should be taken into consideration. In addition, the sample for flow cytometry analysis only needs to extract DNA, and the complete nuclear structure needs to be preserved if the complete DNA morphological structure needs to be preserved, so a kit and a consumable for extracting the complete nuclear are needed, so that the extraction of the nuclear in the paraffin-embedded tissue sample can be quickly and smoothly completed.

SUMMERY OF THE UTILITY MODEL

In order to achieve the purpose, the utility model provides a paraffin-embedded tissue sample cell nucleus extraction kit which comprises a kit body, a proteolytic enzyme reagent bottle, a buffer solution reagent bottle, an eluent A reagent bottle, an eluent B reagent bottle, an eluent C reagent bottle, an eluent D reagent bottle, a fixed solution reagent bottle and a fixing part with seven clamping grooves, wherein the fixing part is detachably or non-detachably arranged in the kit body, and the seven reagent bottles are respectively fixed by the seven clamping grooves.

Further, the eluent A is dimethylbenzene, the eluent B is absolute ethyl alcohol or an ethanol solution with the concentration of more than 85%, the eluent C is an ethanol solution with the concentration of 65% -84%, the eluent D is distilled water, and the fixing liquid is formalin.

The ethanol solution is an ethanol water solution, and the percentage concentration of the ethanol water solution is the volume percentage of ethanol.

Preferably, the proteolytic enzyme reagent bottle and the eluent A reagent bottle are brown glass reagent bottles, and the other reagent bottles are white or milky white plastic reagent bottles.

Further, the proteolytic enzyme was stored at-20. + -. 5 ℃.

Preferably, the proteolytic enzyme is subtilisin, the cell buffer is PBS, and the formalin is 3-6% formaldehyde solution.

Preferably, the capacity of the proteolytic enzyme reagent bottle is the smallest, the capacity of the buffer reagent bottle is the largest, the capacities of the eluent B, the eluent C and the eluent D reagent bottles are equal, the capacity of the eluent A reagent bottle is twice of the capacities of the other eluent reagent bottles, and the capacity of the fixed liquid reagent bottle is larger than the capacity of the eluent B reagent bottle.

In some embodiments, the clamping grooves of the buffer solution reagent bottles are arranged in one row and the clamping grooves of the other six reagent bottles are arranged in two rows and three columns; preferably, the clamping grooves of the protease reagent bottle and the eluent A reagent bottle are arranged in one row, and the clamping grooves of the eluent B reagent bottle, the eluent C reagent bottle, the eluent D reagent bottle and the stationary liquid reagent bottle are divided into two rows and two columns.

In a further embodiment, one side of the clamping groove of the protease reagent bottle and the clamping groove of the eluent A reagent bottle is the clamping groove of the buffer solution reagent bottle, and the other side of the clamping groove of the eluent B reagent bottle, the eluent C reagent bottle, the eluent D reagent bottle and the clamping groove of the fixed solution reagent bottle, so that the protease reagent bottle is arranged at a position on the fixed part which is slightly in the middle, an operator can conveniently take the reagent kit and directly take out the protease reagent bottle, the reagent bottle is placed at-20 +/-5 ℃ for storage, and the two glass reagent bottles are placed at the middle position to better avoid collision. Because the buffer solution is the biggest among seven kinds of reagents, consequently with the biggest, buffer solution reagent bottle independent setting of the heaviest of volume, weight, four kinds of reagents dispersion of its another side of correspondence are put, avoid the kit because of the weight inequality, appear empting the phenomenon such as in transportation or storage process, lead to the reagent excessive.

In other embodiments, to further balance the weights of the two sides, the eluent a reagent bottle with the heaviest weight of the eluents and the fixing solution reagent bottle may be arranged in a row on the corresponding side of the buffer reagent bottle, and the other three eluent reagent bottles and the proteolytic enzyme reagent may be arranged in the middle.

Preferably, the reagent bottle is a wide-mouth bottle, namely, after the bottle cap of the reagent bottle is removed, the diameter of the opening is not less than 80% of the diameter of the bottle body, so that the reagent can be poured conveniently.

Preferably, the fixing member is made of a lightweight shock-resistant material for preventing collision and displacement and facilitating taking, and the material may be selected from foam, hollow cardboard, sponge, etc.

In some embodiments, the fixing part is made of brittle and shock-resistant material (such as foam plastic), the fixing part can be detached, the outer contour of the cross section of the fixing part is the same as or slightly smaller than the inner contour of the cross section of the box body (the gap is smaller than 1 mm), and the slightly smaller contour enables the fixing part to be easily placed into the box body but not easily displaced; the diameters of the seven clamping grooves are respectively the same as or slightly larger than the diameters of the corresponding reagent bottles (the gaps are smaller than 1 mm), and the slightly larger diameters enable the reagent bottles to be easily placed into the clamping grooves but not to be easily displaced.

In some embodiments, the fixing part is made of a flexible deformable material (such as sponge), the outer contour of the cross section of the fixing part is the same as or slightly larger than the shape and size of the inner contour of the cross section of the box body (the deformable material completely abuts against the box body but does not deform with naked eyes), the diameters of the seven clamping grooves are respectively the same as or slightly smaller than the diameters of the corresponding reagent bottles (the deformable material completely abuts against the box body but does not deform with naked eyes), and the relative fixing between the box body and the fixing part and between the fixing part and the reagent bottles can be achieved through extrusion force and friction force.

In some embodiments, the height of the fixing part is not more than one third or one fourth of the height of the highest reagent bottle, so that the reagent bottle is convenient to take. In other embodiments, the height of the securing member exceeds two-thirds of the height of the lowest reagent bottle, which makes it less likely that the reagent bottle will fall out of the retaining groove.

Optionally, the retaining groove is a through hole or a groove.

Preferably, the bottom surface in the reagent box is also provided with an anti-seismic bottom mat, and the material of the anti-seismic bottom mat can be the same as or different from that of the fixing part.

Preferably, the fixing member has a double-layer structure.

In some embodiments, the height of the fixing part is 2-5 cm.

In some embodiments, the fixing part is a double-layer structure and is arranged at the bottom in the box body, the upper layer and the lower layer are respectively provided with seven through holes which correspond to each other and are used as clamping grooves, seven reagent bottles can be conveniently and respectively taken out through the design, and the double-layer structure is not easy to fall out of the clamping grooves in the transportation process compared with a single-layer structure (comprising a single-layer structure with the same thickness as the double-layer structure).

In other embodiments, the fixing component is a double-layer structure, the upper layer is provided with seven through holes serving as clamping grooves, the lower layer is provided with seven corresponding grooves serving as clamping grooves, and the lower layer can be used as a bottom pad and also plays a role in fixing the reagent bottle.

As the number of pathological sections required for nuclear analysis and diagnosis in hospitals is large, the kit is convenient to take and meets the requirement on the number of samples as much as possible when the packaging specification is designed, the reagent amount in the kit can be used for extracting 5 parts, 10 parts, 15 parts, 20 parts, 25 parts or 30 parts of pathological section samples, correspondingly, the reagent reserve of the proteolytic enzyme is preferably 20-45 IU, 50-75 IU, 80-105 IU, 110-135 IU, 140-IU 165 or 170-195 IU, the reagent reserve of the buffer solution is preferably 110-130 mL, 230-250 mL, 350-370 mL, 470-490 mL, 590-610 mL or 710 mL, the reagent reserve of the eluent A is preferably 45-55 mL, 90-110 mL, 135-160 mL, 180-210 mL, 225-260 mL or 275-320 mL, the reagent reserve of the eluent B/C/D is preferably 22-28 mL, 45-55 mL, 67-80 mL, 90-105 mL, 112-130 mL or 137-160 mL.

Preferably, the preservation temperature of the reagent kit except for the proteolytic enzyme is 10-30 ℃.

Furthermore, the kit for extracting the cell nucleus of the paraffin-embedded tissue sample further comprises one or more of a screen, filter paper, a magnetic stirrer, black paperboard, a glass bottle, a glass slide, a centrifuge tube and aluminum foil paper; the aperture of the screen is preferably 50-70 μm, and the aperture of the filter paper is preferably 10-30 μm, so that the filter paper can be used for a cell smear centrifuge.

Preferably, the screen is a nylon screen, and the shape of the screen is preferably circular.

Preferably, the inner cross-sectional diameter of the glass bottle is 1-1.5 cm.

Preferably, the length of the magnetic stirrer is 0.2-0.5 cm, and the width of the magnetic stirrer is 0.08-0.2 cm.

Preferably, the number of the centrifugal tubes is twice the number of the glass bottles.

Preferably, the filter paper is circular in shape.

Further, the utility model provides a paraffin-embedded tissue sample cell nucleus extraction kit comprising the kit, and the kit further comprises a consumable box, wherein the bottommost layer of the consumable box is provided with an anti-seismic bottom pad, and one or more of a screen, filter paper, a magnetic stirrer, black paperboard, a glass bottle, a glass slide, a centrifuge tube and aluminum foil paper are placed on the anti-seismic bottom pad.

Preferably, an anti-seismic top pad is further arranged in the consumable box and placed on the upper layer of the content in the consumable box, so that the anti-seismic effect is achieved on one hand, and the content in the box can be prevented from falling out from the opening on the other hand.

In some embodiments, the consumable cartridge includes at least a centrifuge tube, a glass vial, a slide, a screen, filter paper, and a magnetic stirrer. One side of the centrifugal tube close to the box body is stacked on an anti-seismic bottom pad of the consumable box, and the glass bottle is stacked on the centrifugal tube; the centrifuge tube is preferably a 15 mL centrifuge tube, 5 tubes in one pack, at least two packs, and the glass vial is preferably 10 mL capacity, also 5 packs. The screen, the filter paper and the magnetic stirrer are respectively packaged and placed against the other side of the box body, and the glass slide can be stacked on the glass bottle or vertically placed on one side of the screen, the filter paper and the magnetic stirrer.

In further embodiments, a shock-resistant pad is placed on the glass bottle to isolate the glass bottle from the slide.

In still other embodiments, the slides have individual shock resistant packaging which may be a sponge, foam, bubble film bag, or the like.

In still further embodiments, the consumable cartridge has three compartments, one compartment containing centrifuge tubes and glass vials, the middle compartment containing slides, and the other compartment containing a screen, filter paper, and magnetic stirrer.

Further, the kit or the kit of the present invention further comprises instructions for use.

Further, the kit or kit of the present invention may be used in combination with a nuclear staining reagent, particularly for DNA ploidy analysis and/or chromatin conformation analysis in digital image analysis.

Further, the kit or the kit of the present invention is particularly suitable for nuclear scanning analysis of a digital pathology scanning analysis system. Preferably, the digital pathology scan analysis system has a function of analyzing an image by training a machine learning algorithm.

In a preferred embodiment of the utility model, the method of using the kit or kit comprises the following steps:

1) slicing: preparing a paraffin-embedded tissue section with the thickness of 40-60 mu m or a paraffin-embedded tissue section containing 2000-20000 cell nuclei; the slices can not be too thin or too thick, too thin fragments are more, and too thick fragments can not dewax thoroughly; the number of slices is preferably two or more;

2) dewaxing: sequentially soaking and dewaxing or washing by using an eluent A, an eluent B, an eluent C and an eluent D; preferably in a small glass bottle with the cross section diameter of 1-1.5 cm, ensuring that the sample can be immersed in the eluent, reducing the dosage of the eluent as much as possible, and covering the bottle mouth with aluminum foil paper during the processes of soaking, dewaxing and washing for incubation for a period of time; the eluent A is dimethylbenzene, and the times of soaking and dewaxing by the eluent A are at least two times; eluting B and C with ethanol solution, wherein the concentration of eluent B is higher than that of eluent C, eluting D with distilled water, sequentially washing with eluent B, C, D after dewaxing eluent A, sucking out the former eluent after each washing, and adding the latter eluent; wherein, the eluent D is added in two times;

3) enzyme digestion: digesting the cell nucleus with a proteolytic enzyme; the preferable digestion temperature is 22-27 ℃, and the digestion time is 60-90 minutes; the enzyme is a protease lysate which is prepared freshly and has the concentration of 3-8 IU/mL; storing the protease lysate in an environment of 37 +/-1 ℃ before digestion;

4) and (3) filtering: filtering with a screen with the aperture of 50-70 mu m to remove impurities; centrifuging after filtering, collecting precipitate, preferably observing tissues in a centrifuge tube through a black color comparison card in the whole process, and ensuring that the tissues are not lost; then resuspending the pellet with buffer;

5) cell centrifugal smear: preparing a cell nucleus single-layer smear with the cell nucleus number of 1000-1500 by using a cell smear centrifuge; the cells are preferably spaced 1-3 cells apart under a microscopic high power field. Good single layer nuclei, nuclei should not overlap;

6) fixing: fixing with fixing liquid.

The paraffin-embedded tissue sample cell extraction kit is used for preprocessing a paraffin-embedded tissue section sample, so that cell nucleuses in the sample are released from tissues and cells. So as to facilitate the detection of DNA substances in paraffin-embedded tissue cell nuclei using in vitro diagnostic reagents or instruments. The release of cell nucleuses in paraffin-embedded tissue samples is carried out by dewaxing, rinsing and digesting, eluting and releasing paraffin, cytoplasm and protein, purifying to obtain single cell nucleuses, spreading the single cell nucleuses on a glass slide in a cell nucleus monolayer mode, and finally obtaining a smear which can be used for scanning to form a cell nucleus monolayer image and then analyzing.

The paraffin-embedded tissue sample cell extraction kit is simple in structure, reasonable in layout, convenient to take and use, the fixing parts can be disassembled, the reagent bottles are not easy to separate and shift, the packaging specification is determined according to the use habit of the pathology department of a hospital, the simultaneous operation of a large number of samples is facilitated, the kit and consumable parts are also provided, and a doctor can select the kit according to actual needs.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a schematic structural view of the kit described in example 1;

FIG. 2 is a schematic front view of the fixing member and the reagent bottle of example 2;

FIG. 3 is a schematic front view of the consumable cartridge according to example 3;

FIG. 4 is a schematic front view of the consumable cartridge according to example 4;

1-kit box body, 2-reagent bottle, 21-buffer solution reagent bottle, 22-proteolytic enzyme reagent bottle, 23-eluent A reagent bottle, 24-eluent B reagent bottle, 25-eluent C reagent bottle, 26-eluent D reagent bottle, 27-stationary liquid reagent bottle, 3-fixing part, 31-clamping groove, 32-upper fixing part, 33-lower fixing part, 4-consumable box body, 41-anti-seismic bottom pad, 42-anti-seismic top pad, 43-partition plate, 44-anti-seismic pad, 51-centrifuge tube, 52-glass bottle, 53-glass slide, 531-anti-seismic package, 54-black paperboard, 55-nylon screen, 56-filter paper and 57-magnetic stirrer.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the utility model and are not to be construed as limiting the utility model.

In the description of the present invention, it is to be understood that the terms "middle", "cross section", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or component being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be considered as limiting the present invention. "plurality" means two or more, and "plurality" means two or more, unless specifically limited otherwise.

Example 1

Fig. 1 shows an embodiment of the present invention, and as shown in the figure, the kit for extracting cell nuclei from paraffin-embedded tissue samples comprises a box body 1, reagent bottles 2 and a fixing part 3, wherein a clamping groove 31 is arranged in the fixing part 3, the clamping groove is seven circular through holes arranged in the fixing part 3, wherein seven reagent bottles are arranged, the first reagent bottle 21 on the left side is the largest and is a cell buffer reagent bottle, the cell buffer reagent bottle is arranged in a row, two reagent bottles are arranged in a row close to the reagent bottle 21, and are respectively a protease reagent bottle 22 and an eluent a reagent bottle 23, which are brown glass bottles, wherein the protease reagent bottle 22 is the smallest and is arranged at a position close to the middle, so that an operator can take out the reagent bottles and put the reagent bottles into a refrigerator at-20 ± 5 ℃. The eluent B reagent bottle 24 and the eluent C reagent bottle 25 are arranged in the right column, the eluent D reagent bottle 26 and the stationary liquid reagent bottle 27 are arranged in the rightmost column, and since a plurality of smears can be made on one sample, the using amount of the stationary liquid is larger than that of the eluent, the stationary liquid reagent bottle 27 is also larger than that of the eluent reagent bottle. In the figure 1, the reagent bottles are put together for convenience of taking out in sequence when in use and the fixing liquid is used finally, so that the reagent bottles are put on the rightmost side. The kit of this embodiment can be used for 5 samples, and the preferred reserves of reagents in each reagent bottle are: 120 mL of buffer solution, 30 IU of proteolytic enzyme, 50 mL of eluent A, 25 mL of eluent B, 25 mL of eluent C, 25 mL of eluent D and 50 mL of stationary liquid.

The fixing part 3 can be detachably or non-detachably arranged in the box body 1 and is made of foam plastic, the outer contour of the fixing part is slightly smaller than the inner contour of the box body, the fixing part can be conveniently placed in or taken out during detachment, the fixing part can hardly move in the horizontal direction, the diameter of the clamping grooves of the seven reagent bottles is slightly larger than that of the reagent bottles, and the height of the fixing part is 2-5 cm, so that the reagent bottles are convenient to take and are not easy to drop out of the clamping grooves. Seven reagent bottles are wide-mouth bottles and are convenient to pour.

Example 2

Another embodiment of the fixing component of the present invention is shown in fig. 2, wherein the fixing component 3 is composed of an upper layer and a lower layer, wherein the clamping groove in the upper layer 32 is a circular through hole, the clamping groove in the lower layer 33 is a circular groove, the lower layer can play a role in fixing and anti-seismic, and can be regarded as a bottom pad, and the fixing component has two layers: if meet in the transportation and beat, the lower floor can be combatted earthquake, and the upper strata can take place ascending displacement along with the reagent bottle together, but because the effect in screens groove makes and does not take place the displacement between the reagent bottle, gets back to original position more easily when falling back. The total height of the upper and lower layers of fixing parts is 3-5 cm, and in other embodiments, the bottom pad can be provided with no clamping groove.

Example 3

FIG. 3 shows a preferred embodiment of the kit of the utility model, wherein the box body 4 of the consumable box is provided with an anti-seismic bottom pad 41 and an anti-seismic top pad 42, the box body is divided into two spaces by a partition 43, the space slightly larger on the right side in the figure is used for placing 15 mL centrifuge tubes 51, 10 mL glass bottles 52 and cover glass 53, and they are packaged by five or ten packaging units, as shown in the figure, two layers of centrifuge tubes 51 are placed on the bottom pad 41, 10 centrifuge tubes are arranged on the centrifuge tubes, one layer of anti-seismic pad 44 is arranged on the centrifuge tubes, one layer of glass bottles 52 is placed on the anti-seismic pad, 5 glass bottles are placed on the centrifuge tubes, a second layer of anti-seismic pad 44 is placed on the centrifuge tubes, the top layer is glass slide 53, 10 or 5 sheets of glass are stacked together, two sheets are stacked together, and a packaging bag 531 is wrapped outside the glass slide 53, the anti-seismic packaging bag can be sponge, foam plastic, bubble film packaging bag and the like, the anti-seismic package is mainly used for preventing glass slides made of glass materials which are overlapped together from being cracked when in collision, and also can prevent the glass slides from colliding with glass bottles made of glass materials, and the glass materials of the glass slides and the glass bottles can be borosilicate glass. Additionally, in some embodiments, either of shock resistant packaging 531 and shock resistant pad 44 may be used. In other embodiments, a glass bottle is placed on the first layer of anti-seismic bottom pad 41, a centrifuge tube 51 made of polypropylene is placed on the first layer of anti-seismic bottom pad 44, and a glass slide 53 is placed on the second layer of anti-seismic bottom pad 44, so that the consumables made of two glass materials are separated from each other to avoid collision.

The slightly smaller space on the left side in fig. 3 is used for placing black cardboard 54, nylon screen 55, filter paper 56 and magnetic stirrers 57, the number of the black cardboard is 2, the number of the nylon screen is 5, the number of the filter paper is 10, the number of the magnetic stirrers is 10, and each consumable can be packaged in a plastic bag respectively. The aperture of the nylon screen is 50-70 μm, and the aperture of the filter paper is 10-30 μm. The antidetonation top pad in this embodiment is used for the anticollision, is used for preventing that the consumptive material from falling out in the gap of box mouth simultaneously.

Example 4

As shown in fig. 4, the bottom pad 41 in this embodiment is the same as the top pad 42 and in embodiment 3 except that the case is partitioned into three spaces by the partition plates 43. The larger space on the right is used to store centrifuge tube 51 and glass vial 52, separated by shock-resistant pad 44, which shock-resistant pad 44 may be omitted in some embodiments. The left smaller space is used for storing black cardboard 54, nylon screen 55, filter paper 56 and magnetic stirrer 57, and the consumables are not fragile products and can be randomly placed, and the positions in the drawing are only schematic. The minimum space in the middle is used for placing the glass slide 53, the size of the space is similar to that of the glass slide 53, the glass slide is not easy to move when placed in the space, and the anti-seismic packing material can be packaged again or can be directly placed without being packaged.

Example 5 extraction of nuclei from Paraffin-Embedded tissue samples

The kit used in this example contains:

lyase (subtilisin) 3 mg;

120 mL of cell buffer solution (PBS);

eluent A (dimethylbenzene) 50 mL;

eluent B (95% ethanol) 25 mL;

eluent C (80% ethanol) 25 mL;

eluent D (distilled water) 25 mL;

50 mL of a fixing solution (4% aqueous formaldehyde solution).

The amount of the reagent in the kit can be used for extracting cell nuclei from 5 samples. The kit can be transported at 2-8 deg.C, the lyase is stored at-20 + -5 deg.C, other reagents are stored at normal temperature, and the effective period of the kit is 18 months.

The kit may further comprise consumables: 5 nylon cell screens (aperture 60 μm), 5 filter papers (aperture 15 μm), 10 magnetic stirrers, 2 black cardboard, 5 borosilicate glass vials (10 mL), 10 borosilicate glass slides, and 10 centrifuge tubes (15 mL).

The steps of extracting the cell nucleus are as follows:

first, slicing

The paraffin embedded tissue section with the thickness of 50 mu m is prepared into 2 slices, the slice cannot be too thin or too thick, the too thin slice has more fragments, and the dewaxing is not easy to be thorough if the too thick slice is too thick.

Second, dewaxing

2.1 Each sample was placed in a 10 mL glass vial, 4 mL of eluent A was added to each sample using an electron pipettor, and the tissue was thoroughly infiltrated by gentle shaking. Incubate for 15 minutes with the mouth of the bottle covered with aluminum foil.

2.2 remove the eluent A with a rubber-tipped dropper, the action should be light, avoid sucking out the tissue.

2.3 repeat adding 4 mL of eluent A to each sample with an electron pipette and gently shaking to allow the tissue to infiltrate well. Incubate for 15 minutes with the vial mouth covered with aluminum foil paper, then remove eluent a with a dropper.

2.4 Add 4 mL of eluent B with a continuous applicator, rotate the glass vial to thoroughly infiltrate the tissue, and incubate for 5 minutes.

2.5 removal of eluent B using a separate pipette per sample.

Third, fluid infusion and rinsing

3.1 Add 4 mL of eluent C accurately with a continuous applicator and incubate for 5 min.

3.2 after changing the pipette tip, accurately adding 2 mL of eluent D by using a continuous sample injector, adding 2 mL of eluent D again at an interval of 2 minutes, and waiting for 2 minutes.

3.3 pour the contents of the vial into a 15 mL centrifuge tube, ensure no tissue remains in the vial and the tissue is at the bottom of the centrifuge tube.

3.4 adjust centrifuge to slow down and set as "3", 1500 rpm/min centrifugation 10 minutes, set up the tissue in the centrifuge tube with black color comparison card, gently abandon the supernatant, the dry water is inhaled on the back-off paper, the tissue in the centrifuge tube is observed through black color comparison card in the whole course, ensures that the tissue does not run off.

3.5 Add 8 mL of cell buffer with a continuous applicator. Ensure that the tissue settles at the bottom of the centrifuge tube. Centrifuge deceleration was adjusted to "3", 1500 rpm/min for 10 minutes. The tissue is backed by a black colorimetric card, the supernatant is poured out firstly to ensure that the tissue does not run off, then a glass dropper is used for carefully sucking the residual supernatant, and water is sucked on a water absorption paper in a reversed way.

Fourthly, enzyme digestion and cell nucleus extraction

4.1 taking out the lyase, adding 6 mL of cell buffer solution, mixing uniformly, preparing 0.5 mg/mL lyase solution, and standing at room temperature.

4.2 the prepared 0.5 mg/mL lyase solution is put into a constant-temperature 37 ℃ water bath box for preheating for standby.

4.3 to a 15 mL centrifuge tube, 1 mL of a 0.5 mg/mL solution of the lyase enzyme was added. 1-2 magnetic stirrers were then added to the sample. Digesting for 1 hour and 15 minutes on a magnetic stirrer at room temperature (25 ℃), waiting for 5 minutes and 10 minutes at most if undigested, and mixing for 10 seconds by using a vortex apparatus after digestion is finished.

4.4 Add 8 mL of cell buffer pre-cooled at 4 ℃ to stop enzyme activity.

4.5A new 15 mL centrifuge tube was taken, the 60 μm mesh was symmetrically folded 2 times, and the cone shape was inserted into the new tube. The filtered sample was filtered through a 60 μm screen into a new tube.

4.6 adjust centrifuge to slow down and set as "3", 2500 rpm/min centrifugation 20 minutes, set up the tissue in the centrifuge tube with black color comparison card, gently abandon the supernatant, the dry water is absorbed on the back-off paper, the tissue in the centrifuge tube is observed through black color comparison card in the whole course, ensures that the tissue does not run off.

4.7 to a 15 mL centrifuge tube, 0.5-3 mL of cell buffer was added, depending on the size of the particles. Vibrating and blowing uniformly.

Fifthly, preparing a cell nucleus single-layer glass slide

5.1 marking the serial numbers of the filter paper and the glass slide, firstly putting the glass slide into a centrifugal clamp, then putting the filter paper, and finally putting the glass slide into a funnel for clamping. 100 μ L of the nuclear suspension was added to the funnel and the cell smear centrifuge was adjusted to "low", 600 rpm/min for 5 minutes. When centrifugation is complete, the slide is removed.

5.2 microscopic examination. The amount of suspension is increased or decreased appropriately according to the number of cells and the smear is performed again. The cells are preferably spaced 1-3 cells apart under a microscopic high power field. Good single layer nuclei, nuclei should not overlap.

5.3 fixation with fixative (reagents need to be soaked over the nuclear monolayer) for at least 15 minutes after microscopic examination of the nuclear monolayer slide.

The foregoing detailed description of the preferred embodiments of the utility model has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims (12)

1. The paraffin-embedded tissue sample cell nucleus extraction kit is characterized by comprising a kit body, a proteolytic enzyme reagent bottle, a buffer solution reagent bottle, an eluent A reagent bottle, an eluent B reagent bottle, an eluent C reagent bottle, an eluent D reagent bottle, a fixed solution reagent bottle and a fixing part with seven clamping grooves, wherein the fixing part is detachably or non-detachably arranged in the kit body, and the seven reagent bottles are respectively fixed by the seven clamping grooves.

2. The kit for extracting nuclei of paraffin-embedded tissue sample according to claim 1, wherein the eluent A is xylene, the eluent B is absolute ethanol or an ethanol solution of more than 85%, the eluent C is an ethanol solution of 65% to 84%, the eluent D is distilled water, and the fixing solution is formalin.

3. The paraffin-embedded tissue sample cell nucleus extraction kit as claimed in claim 1, wherein the clamping grooves of the buffer solution reagent bottles are arranged in one row and the clamping grooves of the other six reagent bottles are arranged in two rows and three columns.

4. The paraffin-embedded tissue sample cell nucleus extraction kit as claimed in claim 1, wherein one side of the clamping groove of the proteolytic enzyme reagent bottle and the clamping groove of the eluent A reagent bottle is the clamping groove of the buffer solution reagent bottle, and the other side is the clamping groove of the eluent B reagent bottle, the eluent C reagent bottle, the eluent D reagent bottle and the fixing solution reagent bottle.

5. The paraffin-embedded tissue sample cell nucleus extraction kit as claimed in claim 1, wherein the fixing member is made of a light shock-resistant material.

6. The paraffin-embedded tissue sample cell nucleus extraction kit of claim 1, further comprising one or more of a screen, a filter paper, a magnetic stirrer, black cardboard, a glass bottle, a glass slide, a centrifuge tube, and aluminum foil.

7. A paraffin-embedded tissue sample cell nucleus extraction kit comprising the paraffin-embedded tissue sample cell nucleus extraction kit according to any one of claims 1 to 6, further comprising a consumable box, wherein the bottommost layer of the consumable box is provided with an anti-seismic bottom pad on which one or more of a screen, filter paper, a magnetic stirrer, black cardboard, a glass bottle, a glass slide, a centrifuge tube and aluminum foil paper are placed.

8. The paraffin-embedded tissue sample cell nucleus extraction kit of claim 7, wherein a shock-resistant top pad is further disposed in the consumable box and placed on an upper layer of contents in the consumable box.

9. The paraffin-embedded tissue sample cell nucleus extraction kit of claim 7, wherein the consumable box comprises at least a centrifuge tube, a glass bottle, a glass slide, a screen, filter paper and a magnetic stirrer, wherein the centrifuge tube is stacked on the shock-resistant bottom pad of the consumable box by one side of the box body, the glass bottle is stacked on the centrifuge tube, the glass slide is stacked on the glass bottle, and the screen, the filter paper and the magnetic stirrer are respectively packaged and placed by the other side of the box body.

10. The paraffin-embedded tissue sample cell nucleus extraction kit of claim 9, wherein the glass bottle is further paved with a shock-resistant pad.

11. The paraffin-embedded tissue sample cell nucleus extraction kit of claim 9, wherein the slides have individual shock resistant packaging.

12. The paraffin-embedded tissue sample cell nucleus extraction kit according to claim 7, wherein the consumable box comprises at least a centrifuge tube, a glass bottle, a glass slide, a screen, a filter paper and a magnetic stirrer, and three compartments are arranged in the consumable box, wherein the centrifuge tube and the glass bottle are placed in the compartment on one side, the glass slide is placed in the compartment in the middle, and the screen, the filter paper and the magnetic stirrer are placed in the compartment on the other side.

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