Preparation method of dyeing sample

Abstract

The invention discloses a preparation method of a dyed sample, which comprises the following steps: (S10) passing a biological sample containing a to-be-dyed label through a non-epoxy microporous membrane, and collecting the to-be-dyed label on the non-epoxy microporous membrane; (S20) fixing the to-be-dyed label on the non-epoxy microporous membrane by using a fixing agent at a fixed time, wherein the fixed time is greater than or equal to 8 hours; the time for fixing the to-be-dyed label is greatly prolonged, and the dye is filtered in advance, so that the to-be-dyed label can be stably fixed on the non-epoxy microporous membrane, and the dye is filtered in advance, so that the influence of dye crystals and impurities in the dye on observation is avoided; the problems that a to-be-dyed label cannot be effectively fixed on a non-epoxy microporous membrane by using a conventional chemical dyeing technology, and circulating tumor cells on the non-epoxy microporous membrane are difficult to successfully dye are solved.

Description

Technical Field

[0001] This invention relates to the field of stained sample preparation technology, and in particular to a method for preparing stained samples. Background Technology

[0002] Circulating tumor cells (CTCs) are tumor cells that have entered the bloodstream from the site of tumor growth. CTCs may then travel through the bloodstream to other organs, causing metastasis and resulting in metastatic cancer.

[0003] Currently, there are many methods for testing circulating tumor cells (CTCs) to help determine the risk of cancer recurrence or metastasis. A common current testing method involves first collecting CTCs from the blood, and then labeling and identifying them using immunofluorescent staining assays.

[0004] While immunofluorescence staining analysis exhibits high specificity for specific biomarkers on circulating tumor cells, it suffers from drawbacks such as high cost and difficulty in sample preservation. Specifically, immunofluorescence staining analysis requires a fluorescence microscope and fluorescent antibodies, thus the cost inevitably includes the purchase of these two items, both of which are very expensive. A fluorescence microscope alone costs approximately NT$1 million, so most laboratories do not typically purchase one. Furthermore, fluorescent antibodies must be refrigerated before use to prevent fluorescence degradation, adding to the cost of antibody preservation. In addition, the fluorescence of fluorescently stained samples obtained after immunofluorescence staining analysis is easily degraded, making effective sample preservation difficult.

[0005] In contrast, chemical staining techniques avoid the aforementioned problems. Specifically, chemical staining uses optical microscopes and chemical stains. The price of an optical microscope (approximately NT$100,000 to NT$300,000) is much lower than that of a fluorescence microscope, so most laboratories have their own optical microscopes. Furthermore, the cost of purchasing chemical stains is lower than that of purchasing fluorescent antibodies, and chemical stains can generally be stored at room temperature and are less prone to degradation. In addition, chemically stained samples prepared using chemical staining techniques are relatively resistant to degradation, thus offering the advantage of long-term preservation.

[0006] However, if non-epoxy microporous membranes are used to collect circulating tumor cells, it is not easy to fix the circulating tumor cells onto the non-epoxy microporous membrane using conventional chemical staining techniques, nor is it easy to successfully stain the circulating tumor cells on the non-epoxy microporous membrane. As a result, it is not possible to clearly identify the staining of the cytoplasm and the cell nucleus under an optical microscope. This is a problem that needs to be solved. Summary of the Invention

[0007] In view of this, the present invention addresses the deficiencies of the prior art, and its main objective is to provide a method for preparing stained samples that effectively solves the problem that existing conventional chemical staining techniques cannot effectively fix the target to be stained onto non-epoxy microporous membranes, and it is difficult to successfully stain circulating tumor cells on non-epoxy microporous membranes.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a stained sample includes the following steps:

[0010] (S10) Pass a biological sample containing a target to be stained through a non-epoxy microporous membrane to collect the target to be stained on the non-epoxy microporous membrane.

[0011] (S20) The target to be stained is fixed on the non-epoxy microporous membrane with a fixative for a fixed time, wherein the fixation time is greater than or equal to 8 hours; and

[0012] (S30) The target to be stained on the non-epoxy microporous membrane is stained using at least one filtered dye.

[0013] As a preferred embodiment, the fixed time is 8 to 32 hours.

[0014] As a preferred embodiment, the at least one filtered dye is obtained by filtering at least one dye through a filter membrane.

[0015] As a preferred embodiment, the pore size of the filter membrane is less than or equal to 0.22 μm.

[0016] As a preferred embodiment, the fixative comprises methanol, ethanol, denatured ethanol, propanol, isopropanol, diethyl ether, formaldehyde, acetone, acetic acid, urea, chloroform, hydrogen chloride, polyethylene glycol, or combinations thereof.

[0017] As a preferred embodiment, the fixative is 100% (v / v) methanol, 50% (v / v) ethanol, 95% (v / v) ethanol, 100% (v / v) ethanol, 95% (v / v) denatured ethanol, 80% (v / v) propanol, 80% (v / v) isopropanol, ethanol-ether fixative (ethanol volume: ether volume = 1:1), 4% (v / v) formaldehyde fixative, acetone fixative, 5% (v / v) glacial acetic acid fixative, 2 mol / L urea, carnoy solution, hydrogen chloride-soluble fixative, polyethylene glycol fixative, or a combination thereof.

[0018] As a preferred embodiment, the at least one dye includes hematoxylin, orange yellow G, eosin Y, brilliant green, bismuth brown, or a combination thereof.

[0019] As a preferred embodiment, the at least one dye includes hematoxylin, orange yellow G, eosin Y, brilliant green, and Bismarck brown.

[0020] As a preferred embodiment, the material of the non-epoxy microporous membrane is a transparent polymer material.

[0021] As a preferred embodiment, the transparent polymer material is polyimide, polycarbonate, polyethylene, polyethylene terephthalate, polyethylene naphthalate, polymethyl methacrylate, polystyrene, or a combination thereof.

[0022] As a preferred embodiment, after step (S20), a solution for making the biological sample transparent is added to the non-epoxy microporous membrane, and the non-epoxy microporous membrane is sandwiched between a carrier plate and a cover plate.

[0023] As a preferred embodiment, the cover plate is a cover glass sheet, and the cover plate has a thickness of 0.12 mm to 0.17 mm.

[0024] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, as can be seen from the above technical solution:

[0025] By significantly extending the fixation time of the target to be stained and filtering the dye in advance, the target to be stained can be stably fixed on the non-epoxy microporous membrane. Furthermore, by filtering the dye in advance, the influence of dye crystallization and impurities on observation can be avoided. This solves the problem that conventional chemical staining techniques cannot effectively fix the target to be stained on the non-epoxy microporous membrane and are difficult to successfully stain circulating tumor cells on the non-epoxy microporous membrane. Attached Figure Description

[0026] Figure 1 This is a flowchart of the method for preparing stained samples according to the present invention;

[0027] Figure 2 This is a staining result diagram of Comparative Example 1;

[0028] Figure 3 This is a staining result image of Comparative Example 2;

[0029] Figure 4 This is a staining result image of Comparative Example 3;

[0030] Figure 5 This is a staining result diagram of Example 1 of the present invention;

[0031] Figure 6 This is a staining result diagram of Example 2 of the present invention.

[0032] Explanation of reference numerals in the attached diagram:

[0033] 10. Non-epoxy microporous membrane; 12. Micropores

[0034] 20. Dye crystals; 30. Cytoplasm

[0035] 40. Cell nucleus; 50. Nucleolus

[0036] 100. Preparation method of stained samples

[0037] S10, S20, S30, S40, Steps. Detailed Implementation

[0038] This invention discloses a method for preparing stained samples. This method immobilizes the target specimen from a biological sample collected on a non-epoxy-based microporous membrane, ensuring successful staining and allowing clear identification of cytoplasmic and nuclear staining under an optical microscope. Because the fixation time in the fixation step is significantly longer than typical fixation times (e.g., 15-20 minutes), the target specimen is firmly fixed to the non-epoxy-based microporous membrane, preventing detachment during staining. Furthermore, the dyes used in the staining step of this method are filtered to remove dye crystals, thus preventing contamination of the stained sample by dye crystals.

[0039] In this invention, the term "about" refers to a numerical range that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated value in any direction (greater or less).

[0040] like Figure 1The diagram shown is a flowchart illustrating a method 100 for preparing a stained sample according to some embodiments of the present invention. The method 100 for preparing a stained sample includes steps S10, S20, and S30.

[0041] like Figure 1 As shown, in one embodiment, step S10 involves passing a biological sample containing the target to be stained through a non-epoxy-based microfilm to collect the target to be stained onto the non-epoxy-based microfilm.

[0042] In some embodiments, examples of the aforementioned biological samples may include whole blood, serum, plasma, pleural effusion, ascites, synovial fluid, cerebrospinal fluid, semen, urine, feces, saliva, nasopharyngeal swabs, sputum, throat swabs, bronchial lavage fluid (BA), bronchoalveolar lavage fluid (BL), bronchial brush (BB), amniotic fluid, breast milk, sweat, tears, gastric juice, pus, biopsy, etc., but are not limited thereto. In a specific embodiment, the biological sample may be whole blood. In some embodiments, the staining target is cells in the biological sample, particularly tumor cells in the biological sample. Examples of the aforementioned staining target may include, but are not limited to, circulating tumor cells (CTCs) in biological samples such as whole blood, serum, plasma, or urine, wherein the size of the staining target is larger than the size of the micropores on the non-epoxy microporous membrane. In a specific embodiment, the staining target is tumor cells in whole blood.

[0043] In some embodiments, the non-epoxy microporous membrane is a non-epoxy membrane containing multiple micropores. The micropores penetrate the non-epoxy microporous membrane, and the pore size is designed to retain the target on the non-epoxy microporous membrane, for example, micropores with an average diameter less than or equal to 10 μm. In some embodiments, step S10 uses pressure to force the biological sample containing the target to be stained through the non-epoxy microporous membrane, thereby intercepting the target in the biological sample whose volume is larger than the micropores. In a specific embodiment, step S10 involves filtering the biological sample containing the target to be stained using a microfilter containing a non-epoxy microporous membrane (Taiwan Patent Publication No.: I769544) proposed by the applicant of this invention. A pressure difference forces the biological sample through the non-epoxy microporous membrane, causing the target in the biological sample whose volume is larger than the micropores to be intercepted by the non-epoxy microporous membrane, while allowing other components in the biological sample whose volume is smaller than the micropores to flow out through the micropores.

[0044] In some embodiments, examples of materials for the non-epoxy microporous membrane may include transparent polymer materials, such as polyimide, polycarbonate, polyethylene, polyethylene terephthalate, polyethylene naphthalate, polymethyl methacrylate, polystyrene, or combinations thereof, but not limited thereto. In a particular embodiment, the material of the non-epoxy microporous membrane is polyethylene terephthalate. In some embodiments, the shape of the micropores on the non-epoxy microporous membrane may be rectangular, hexagonal, circular, or other shapes, but is not limited thereto. In some embodiments, the number of micropores on the non-epoxy microporous membrane may be greater than 50,000. In some embodiments, the distance between two micropores of the non-epoxy microporous membrane is at least 15 μm. In some embodiments, the thickness of the non-epoxy microporous membrane is from 5 μm to 25 μm. In some embodiments, the non-epoxy microporous membrane is formed by laser processing of the non-epoxy membrane, thereby creating micropores on the non-epoxy membrane, and the micropores penetrate the non-epoxy membrane.

[0045] like Figure 1As shown, in one embodiment, step S20 involves fixing the target to be stained onto a non-epoxy microporous membrane with a fixative at a fixed time. In some embodiments, the fixative may contain methanol, ethanol, denatured ethanol, propanol, isopropanol, diethyl ether, formaldehyde, acetone, acetic acid, urea, chloroform, hydrogen chloride, polyethylene glycol, or combinations thereof, but is not limited thereto. In some embodiments, the concentration of methanol may be about 80% to about 100%, for example, 100% (v / v) methanol, but is not limited thereto. In some embodiments, the ethanol may be about 45% to 100% (v / v) ethanol, for example, 50% (v / v) ethanol, 95% (v / v) ethanol, 100% (v / v) ethanol, but is not limited thereto. In some embodiments, the concentration of denatured ethanol may be about 30% to about 100%, for example, 95% (v / v) denatured ethanol, but is not limited thereto. In some embodiments, the concentration of propanol may be from about 60% (v / v) to about 100% (v / v), for example, 80% (v / v) propanol, but is not limited thereto. In some embodiments, the concentration of isopropanol may be from about 60% (v / v) to about 100% (v / v), for example, 80% (v / v) isopropanol, but is not limited thereto. In some embodiments, ethanol and diethyl ether may be mixed, with an ethanol volume: ether volume ratio of about 1:1 to about 3:1, for example, ethanol volume: ether volume = 1:1, but is not limited thereto. In some embodiments, the concentration of formaldehyde may be from about 4% (v / v) to about 40% (v / v), for example, 4% (v / v) formaldehyde, but is not limited thereto. In some embodiments, the concentration of acetic acid may be from about 0.5% (v / v) to about 10% (v / v), for example, about 1% (v / v) acetic acid, the source of which may be glacial acetic acid, but is not limited thereto. In some embodiments, the concentration of the urea may be from about 1 mol / L to about 10 mol / L, for example, 2 mol / L urea, but is not limited thereto. In some embodiments, the fixative may be 100% (v / v) methanol, 50% (v / v) ethanol, 95% (v / v) ethanol, 100% (v / v) ethanol, 95% (v / v) denatured ethanol, 80% (v / v) propanol, 80% (v / v) isopropanol, ethanol-ether fixative (ethanol volume: ether volume = 1:1), 4% (v / v) formaldehyde fixative, acetone fixative, 5% (v / v) glacial acetic acid fixative, 2 mol / L urea, Carnoy's solution, hydrogen chloride-soluble fixative, polyethylene glycol fixative, or combinations thereof, but is not limited thereto, wherein the aforementioned fixatives expressed as volume percentages (% (v / v)) all use water as a solvent. The carnoi solution may be composed of ethanol and acetic acid, or it may be composed of ethanol, acetic acid, and chloroform. In a particular embodiment, the fixative is 95% (v / v) ethanol.

[0046] In some embodiments, the fixation time is greater than or equal to 8 hours, preferably 8 to 32 hours, for example, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, or 32 hours, but not limited thereto. If the fixation time is less than 8 hours, the target material to be stained may easily detach from the non-epoxy microporous membrane because it is not stably fixed on the non-epoxy microporous membrane. In a specific embodiment, the fixation time is 24 hours. In some embodiments, step S20 involves immersing the non-epoxy microporous membrane containing the target material to be stained in a fixative for 8 to 32 hours, so that the target material to be stained is fixed on the non-epoxy microporous membrane under the dehydration effect of the fixative.

[0047] like Figure 1 As shown, in one embodiment, step S30 involves staining the target on the non-epoxy microporous membrane with at least one filtered dye. In some embodiments, the dye may include hematoxylin, Orange G, Eosin Y, light green, Bismarck brown, or combinations thereof, but is not limited thereto. In a specific embodiment, the dye includes hematoxylin, Orange G, Eosin Y, light green, and Bismarck brown. Hematoxylin is used to stain the basophilic structures in the target blue-purple, and the basophilic structures may be structures containing nucleic acids in the target, such as ribosomes and chromosomes in the cell nucleus. Orange G is used to stain the keratin in the target orange-yellow. Eosin Y is used to stain the eosinophilic structures in the target pink, such as nucleoli in the target, thus Eosin Y can stain mature surface cells pink. Brilliant green is used to stain cells with high metabolic rates in the target sample, resulting in a green, blue-green, or blue color. Cells with high metabolic rates include intermediate squamous epithelial cells, deep squamous epithelial cells, and columnar cells. Bismarck brown is used to stain components such as acidic mucin. In some embodiments, the filtered dye is obtained by filtering the dye through a filter membrane, thereby removing dye crystals and other impurities larger than the pore size of the filter membrane, but this is not a limitation.

[0048] The dyeing time for the target object can be adjusted as needed, for example, from 30 seconds to 10 minutes, specifically 30 seconds, 45 seconds, 60 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes. In some embodiments, the pore size of the filter membrane is less than or equal to 10.0 μm, such as 10.0 μm, 5.0 μm, 3.0 μm, 1.0 μm, 0.8 μm, 0.45 μm, 0.22 μm, or 0.10 μm, but not limited thereto. In a preferred embodiment, the pore size of the filter membrane is less than or equal to 0.22 μm, thereby more effectively filtering out dye crystals and impurities in the dye. In a specific embodiment, the pore size of the filter membrane is 0.22 μm. In some embodiments, step S30 involves immersing a non-epoxy microporous membrane in at least one dye and removing excess dye using a cleaning solution or destaining solution, thereby staining the target material on the non-epoxy microporous membrane to obtain a stained sample. The cleaning solution can be filtered water, distilled water, double-distilled water, deionized water, reverse osmosis water (RO water), pure water, ultrapure water, etc., but is not limited to these. The cleaning solution can also be a fixative as described above, such as 95% (v / v) ethanol, thus achieving both cleaning and fixation of the target material. The destaining solution can be ethanol containing hydrogen chloride and acetic acid, such as 70% (v / v) ethanol containing 0.5% (v / v) hydrogen chloride. The aforementioned destaining solution, expressed as a volume percentage (% (v / v)), uses water as a solvent. In a specific embodiment, the destaining solution is 70% (v / v) ethanol containing 0.5% (v / v) hydrogen chloride. The time for removing excess dye using cleaning and destaining solutions can be adjusted as needed, for example, from 1 second to 30 minutes, specifically such as 1 second, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes, etc.

[0049] like Figure 1 As shown, in some embodiments, the method 100 for preparing stained samples further includes step S40. Step S40 is, after step S30, adding a solution for clearing the biological sample to a non-epoxy microporous membrane, and sandwiching the non-epoxy microporous membrane between the carrier plate and the cover plate. In some embodiments, the solution for clearing the biological sample may be a clearing agent, specifically xylene, isopropanol, limonene, other xylene-free clearing agents, or combinations thereof, wherein the other xylene-free clearing agents are, for example, the Tissue Clearing / Staining Kit (CytoVista). TMThe solution used to clear biological samples is xylene, including but not limited to tissue clearing kits (such as TissueClearing Kits (Abcam)). In one specific embodiment, the solution used to clear the biological sample is xylene. In some embodiments, the clearing time using the clearing agent can be from 1 minute to 30 minutes, specifically 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes, but is not limited to these. In some embodiments, the carrier plate is a transparent carrier plate, such as a glass slide, but is not limited to this. In some embodiments, the cover plate is a transparent cover plate, but is not limited to this, such as a coverslip. In one specific embodiment, the carrier plate is a glass slide, and the cover plate is a coverslip. The thickness of the cover plate can be any thickness, preferably from 0.12 mm to 0.17 mm, such as 0.13 mm, 0.14 mm, 0.15 mm, 0.16 mm, etc., but is not limited to this. By making the coverslip thicker than 0.12 mm, it gains a certain weight, thus reducing the gap width between the coverslip and the slide when placed on top of the slide. This reduces the rate at which the solution used to clear the biological sample leaks out of the gap, extending the preservation time of the stained sample. In some embodiments, sealing adhesive can be used to seal the gap between the coverslip and the slide, further reducing the rate at which the solution used to clear the biological sample leaks out. In some embodiments, step S40 involves adding the solution used to clear the biological sample onto a non-epoxy microporous membrane to make the stained sample transparent, and then sandwiching the non-epoxy microporous membrane between the carrier and the coverslip to preserve the stained sample.

[0050] The following describes some embodiments and comparative examples of the present invention to illustrate more specifically the effects that can be achieved by the disclosed method 100 for preparing stained samples.

[0051] Example

[0052] [Cell staining experiment]

[0053] Examples and comparative examples are provided below. In Comparative Examples 1-3 and Examples 1-2, the microfilter containing a non-epoxy microporous membrane (Taiwan Patent Publication No.: I769544) previously proposed by the applicant of this invention was used to filter biological samples containing staining targets. The biological sample containing the staining target is forced through the non-epoxy microporous membrane by a pressure difference, causing the staining target in the biological sample with a volume larger than the micropore size to be intercepted by the non-epoxy microporous membrane. 7.5 mL of whole blood was used as an example biological sample, with additional breast cancer cells (cell line: Michigan Cancer Foundation-7; MCF-7) added as the staining sample. Breast cancer cells are generally large in volume and therefore easily intercepted by the non-epoxy microporous membrane. Furthermore, polyethylene terephthalate was used as an example material for the non-epoxy microporous membrane.

[0054] [Comparative Examples 1-3]

[0055] 1. Comparative Example 1

[0056] Comparative Example 1 was prepared by staining the target on a non-epoxy microporous membrane using Papanicolaou (PAP) Red Stain Kit (Cytology Stain); product number: ab150679). The staining method was as described in the Papanicolaou (PAP) Red Stain Kit (Cytology Stain). Specifically, the non-epoxy microporous membrane was first fixed by sequentially immersing it in 95% (v / v) ethanol and 70% (v / v) ethanol for 5 minutes. Then, the non-epoxy microporous membrane was immersed in RO water for 2 minutes. Next, the non-epoxy microporous membrane was immersed in hematoxylin for 5.5 minutes, and then excess dye was washed away with RO water. Next, the non-epoxy microporous membrane was fixed by immersing it in 95% (v / v) ethanol. Next, the non-epoxy microporous membrane was immersed in Orange G-6 for 2.5 minutes. Finally, excess dye was washed away with 100% (v / v) ethanol. Next, the non-epoxy microporous membrane was immersed in EA-50 staining solution containing eosin Y, brilliant green, and Bismarck brown for 3.5 minutes. Excess stain was then washed away with 100% (v / v) ethanol, followed by three rinses with 100% (v / v) ethanol. Finally, the non-epoxy microporous membrane was cleared by immersion in xylene and observed under an optical microscope at 40x magnification. The staining results of Comparative Example 1 are shown in the figure below. Figure 2 As shown, Figure 2 The display shows a scale bar of 20μm.

[0057] like Figure 2As shown, in Comparative Example 1, the time for fixing the target to be stained onto the non-epoxy-based microporous membrane 10 with 95% (v / v) ethanol was only 5 minutes at the beginning. Therefore, after the preparation of Comparative Example 1 was completed, breast cancer cells could not be fixed onto the non-epoxy-based microporous membrane 10, resulting in only the non-epoxy-based microporous membrane 10 and its micropores 12 being observed, but no cells were observed. In addition, although due to Figure 2 This is only a partial view of the photograph, therefore it is not included. Figure 2 Dye crystallization was observed (e.g.) Figure 2 The dye crystals shown are 20), but Comparative Example 1 did not filter the dye beforehand, so in reality... Figure 2 The area shown contains a large amount of dye crystals, which severely affects the observation of cells.

[0058] 2. Comparative Example 2

[0059] Comparative Example 2 involved staining a non-epoxy microporous membrane with Papanicolaou (PAP) Red Stain Kit (Cytology Stain); product number: ab150679). The staining method followed the procedure described in the Papanicolaou (PAP) Red Stain Kit. Specifically, the non-epoxy microporous membrane was first immersed in 95% (v / v) ethanol for 15 minutes, then immersed in 70% (v / v) ethanol for 5 minutes for fixation. Next, the membrane was immersed in RO water for 2 minutes. Then, the membrane was immersed in hematoxylin for 5.5 minutes, followed by rinsing with RO water to remove excess stain. Next, the membrane was immersed in 95% (v / v) ethanol for fixation. Finally, the membrane was immersed in Orange G-6 for 2.5 minutes. Next, excess stain was washed away with 100% (v / v) ethanol. Then, the non-epoxy microporous membrane was immersed in EA-50 staining solution containing eosin Y, brilliant green, and Bismarck brown for 3.5 minutes. Next, excess stain was washed away with 100% (v / v) ethanol, followed by three rinses with 100% (v / v) ethanol. Finally, the non-epoxy microporous membrane was cleared by immersion in xylene and observed under an optical microscope at 40x magnification. Comparative Example 2 was prepared using essentially the same method as Comparative Example 1, except that the initial immersion time of the non-epoxy microporous membrane in 95% (v / v) ethanol was changed from 5 minutes to 15 minutes. The staining results of Comparative Example 2 are shown in the figure below. Figure 3 As shown, Figure 3 The display shows a scale bar of 20μm.

[0060] like Figure 3As shown, although Comparative Example 2 initially extended the fixation time of the target on the non-epoxy microporous membrane 10 with 95% (v / v) ethanol to 15 minutes, the staining results were still unsatisfactory. Since the dye was not filtered beforehand in Comparative Example 2, obvious dye crystals 20 were observed in the field of view in addition to the non-epoxy microporous membrane 10 and its micropores 12, severely affecting cell observation. Furthermore, since the fixation time was only extended to 15 minutes, the cells may not have been firmly fixed to the non-epoxy microporous membrane 10, thus affecting the staining effect. Moreover, because it was impossible to clearly observe both cytoplasmic and nuclear staining simultaneously, it was impossible to identify whether the stained object was a cell or impurities, severely impacting cell observation.

[0061] 3. Comparative Example 3

[0062] Comparative Example 3 involved staining a non-epoxy microporous membrane with Papanicolaou (PAP) Red Stain Kit (Cytology Stain); product number: ab150679). The staining method followed the procedure described in the Papanicolaou (PAP) Red Stain Kit. Specifically, the non-epoxy microporous membrane was first immersed in 95% (v / v) ethanol for 15 minutes, then immersed in 70% (v / v) ethanol for 5 minutes for fixation. Next, the membrane was immersed in RO water for 2 minutes. Then, the membrane was immersed in hematoxylin for 5.5 minutes, followed by rinsing with RO water to remove excess stain. Next, the membrane was immersed in 95% (v / v) ethanol for fixation. Finally, the membrane was immersed in Orange G-6 for 2.5 minutes. Next, excess staining agent was washed away with 100% (v / v) ethanol. Then, the non-epoxy microporous membrane was immersed in EA-50 staining solution containing eosin Y, brilliant green, and Bismarck brown for 3.5 minutes. Next, excess staining agent was washed away with 100% (v / v) ethanol, followed by three rinses with 100% (v / v) ethanol. Finally, the non-epoxy microporous membrane was cleared by immersion in xylene and observed under an optical microscope at 40x magnification. The preparation method of Comparative Example 3 was essentially the same as that of Comparative Example 1, except that the initial immersion time of the non-epoxy microporous membrane in 95% (v / v) ethanol was changed from 5 minutes to 15 minutes, and that the hematoxylin, orange G-6, and EA-50 staining solution were filtered through a 0.22 μm filter membrane before the staining step. The staining results of Comparative Example 3 are shown in the figure below. Figure 4 As shown, Figure 4 The display shows a scale bar of 20μm.

[0063] like Figure 4 As shown, although Comparative Example 3 initially used 95% (v / v) ethanol to fix the target to be stained on the non-epoxy microporous membrane 10 for 15 minutes, the staining results were still unsatisfactory. Although Comparative Example 3 filtered the dye beforehand, thus preventing the staining from being observed in the field of view, the results were still unsatisfactory. Figure 3 The staining crystals 20 in the sample were contaminated. However, since the fixation time in Comparative Example 3 was only extended to 15 minutes, the cells may not have been firmly fixed on the non-epoxy microporous membrane 10, thus affecting the staining effect. Furthermore, since it was not possible to clearly observe the staining of the cytoplasm and the cell nucleus at the same time, it was impossible to identify whether the stained object was a cell or an impurity, which seriously affected the observation of the cells.

[0064] [Examples 1-2]

[0065] Examples 1 and 2 describe the preparation of stained samples independently using the method 100 of the present invention, with the same sequence of steps. Specifically, after the non-epoxy microporous membrane intercepts the target to be stained, it is immersed in 95% (v / v) ethanol for 24 hours. Next, it is immersed in RO water for 2 minutes to remove residual ethanol. Then, it is immersed in hematoxylin (trade name: Gill's Hematoxylin V) for 5.5 minutes. Next, it is immersed in RO water for 3 minutes. Then, it is immersed in a destaining solution for 15 seconds to remove excess dye, wherein the destaining solution is 70% (v / v) ethanol containing 0.5% (v / v) hydrogen chloride. Finally, the non-epoxy microporous membrane is sequentially immersed in RO water and 95% (v / v) ethanol for 2 minutes to remove the destaining solution. Next, the non-epoxy microporous membrane was immersed in Orange G (trade name: Orange G-6) for 2.5 minutes. Then, it was immersed in 95% (v / v) ethanol for 2 minutes to remove excess stain. Next, the non-epoxy microporous membrane was immersed in EA-50 staining solution containing eosin Y, brilliant green, and Bismarck brown for 3.5 minutes. Then, it was immersed in 95% (v / v) ethanol for 4 minutes to remove excess stain. Next, the non-epoxy microporous membrane was immersed in 100% (v / v) ethanol for 7.5 minutes to dehydrate and fix the cells. Finally, the non-epoxy microporous membrane was cleared by immersing it in xylene for 20 minutes and then observed under an optical microscope at 40x magnification. In Examples 1 and 2, the hematoxylin, orange yellow G, and EA-50 staining solution containing eosin Y, brilliant green, and Bismarck brown were all filtered through a filter membrane before staining the specimens to remove dye crystals and impurities. The filter membrane had a pore size of 0.22 μm. The staining result of Example 1 is shown in the figure below. Figure 5 As shown in the figure. The staining results of Example 2 are shown in the figure. Figure 6 As shown, Figure 5 and Figure 6 All of them display a scale bar of 20μm.

[0066] like Figure 5 and Figure 6 As shown, in Examples 1 and 2, the time for fixing the target to be stained on the non-epoxy microporous membrane 10 with 95% (v / v) ethanol was significantly extended to 24 hours at the beginning. In addition, the dye was filtered with a filter membrane before use. Therefore, the cytoplasm 30, nucleus 40 and nucleolus 50 of the cells can be clearly observed in the field of view, so that the morphology of the cells can be clearly identified, which is helpful for the examiners to identify whether the cells are tumor cells based on the morphology of the cells.

[0067] As described above, Comparative Examples 1-3 show that under conventional fixation time, insufficient fixation time makes it difficult for the target to be stained to be stably fixed on the non-epoxy microporous membrane. Furthermore, Comparative Example 2 shows that if the dye is not filtered before staining the target, dye crystals may contaminate the stained sample, affecting its quality. In contrast, in Examples 1-2 prepared according to the staining sample preparation method 100 of this application, sufficient fixation time allows for the observation of a large number of cells in the stained sample. Moreover, in Examples 1-2 prepared according to the staining sample preparation method 100 of this application, because the dye is filtered before staining the target, removing dye crystals and other impurities, the cytoplasm and nucleus of cells can be clearly observed in the stained sample, and even the nucleoli within the nucleus can be clearly observed.

[0068] [Cell Counting Experiment]

[0069] In this experiment, cell counting was performed on the stained samples prepared according to the method of Comparative Example 1 and the stained samples prepared according to the methods of Examples 1 and 2 using an optical microscope. Only cells in which both cytoplasmic staining and the cell nucleus could be clearly observed were counted. The results of the cell counting experiment showed that no cells were observed in the stained samples prepared according to the method of Comparative Example 1, while 2,439 cells were observed in the stained samples prepared according to the methods of Examples 1 and 2.

[0070] As described above, further cell technology experiments confirmed that under conventional fixation times, insufficient fixation resulted in the complete absence of recognizable cells on the epoxy-based microporous membrane. In contrast, the stained samples prepared using the method described in this application, due to sufficient fixation time and prior filtration of the dye, successfully yielded thousands of successfully stained cells.

[0071] In summary, the experimental results above demonstrate that the method for preparing stained samples in this application significantly extends the fixation time of the target to be stained and filters the dye beforehand, thereby enabling the target to be stained to be stably fixed on the non-epoxy microporous membrane. Furthermore, by filtering the dye beforehand, the influence of dye crystallization and impurities on observation is avoided. This solves the problem that conventional chemical staining techniques cannot effectively fix the target to be stained on the non-epoxy microporous membrane and are difficult to successfully stain circulating tumor cells on the non-epoxy microporous membrane.

[0072] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a stained sample, characterized in that: Includes the following steps: (S10) Pass a biological sample containing a target to be stained through a non-epoxy microporous membrane to collect the target to be stained on the non-epoxy microporous membrane. (S20) The target to be stained is fixed on the non-epoxy microporous membrane with a fixative for a fixed time, wherein the fixation time is greater than or equal to 8 hours. as well as (S30) The target to be stained on the non-epoxy microporous membrane is stained using at least one filtered dye.

2. The method for preparing stained samples according to claim 1, characterized in that: The fixed time is 8 to 32 hours.

3. The method for preparing stained samples according to claim 1, characterized in that: The at least one filtered dye is obtained by filtering at least one dye through a filter membrane.

4. The method for preparing stained samples according to claim 3, characterized in that: The pore size of the filter membrane is less than or equal to 0.22 μm.

5. The method for preparing stained samples according to claim 1, characterized in that: The fixative comprises methanol, ethanol, denatured ethanol, propanol, isopropanol, diethyl ether, formaldehyde, acetone, acetic acid, urea, chloroform, hydrogen chloride, polyethylene glycol, or combinations thereof.

6. The method for preparing stained samples according to claim 5, characterized in that: The fixative is 100% (v / v) methanol, 50% (v / v) ethanol, 95% (v / v) ethanol, 100% (v / v) ethanol, 95% (v / v) denatured ethanol, 80% (v / v) propanol, 80% (v / v) isopropanol, ethanol-ether fixative (ethanol volume: ether volume = 1:1), 4% (v / v) formaldehyde fixative, acetone fixative, 5% (v / v) glacial acetic acid fixative, 2 mol / L urea, carnoy solution, hydrogen chloride-soluble fixative, polyethylene glycol fixative, or a combination thereof.

7. The method for preparing stained samples according to claim 3, characterized in that: The at least one dye includes hematoxylin, orange yellow G, eosin Y, brilliant green, Bismarck brown, or a combination thereof.

8. The method for preparing stained samples according to claim 7, characterized in that: The at least one dye includes hematoxylin, orange yellow G, eosin Y, brilliant green, and Bismarck brown.

9. The method for preparing stained samples according to claim 1, characterized in that: The non-epoxy microporous membrane is made of a transparent polymer material.

10. The method for preparing stained samples according to claim 9, characterized in that: The transparent polymer material is polyimide, polycarbonate, polyethylene, polyethylene terephthalate, polyethylene naphthalate, polymethyl methacrylate, polystyrene, or a combination thereof.

11. The method for preparing stained samples according to claim 1, characterized in that: Further includes: After step (S30), a solution for making the biological sample transparent is added to the non-epoxy microporous membrane, and the non-epoxy microporous membrane is sandwiched between a carrier plate and a cover plate.

12. The method for preparing stained samples according to claim 11, characterized in that: The cover plate is a cover glass sheet, and the cover plate has a thickness of 0.12 mm to 0.17 mm.

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