Cover glass coated with dye

Abstract

The invention relates to a cover glass coated with a dye and a use method of the cover glass. The present invention provides an improved method of performing an assay on a glass slide using a cover glass coated on one side with one or more dyes and then staining an end product resulting from one or more reactions on a glass slide. The cover glass faces the glass slide with the coated side and is placed on the glass slide, and then products generated after one or more reactions are completed on the glass slide are dyed. The invention simplifies and accelerates the staining process for a reaction completed on a slide.

Description

TECHNICAL FIELD

[0001] The present invention relates to a cover slip for staining and a method of using the same. The present invention provides an improved method for performing a test on a carrier slide. In particular, the present invention provides a method for detecting sperm DNA fragmentation (SDF) in a semen sample. BACKGROUND

[0002] The integrity of sperm DNA is critical for embryo quality, embryo implantation and embryo development. Causes of sperm DNA fragmentation (SDF) can be extrinsic, such as radiation, environmental pollutants or chemotherapeutics, or intrinsic, such as defective spermatogenesis, sperm apoptosis or oxidative stress. SDF can lead to male infertility, failure of in vitro fertilization or miscarriage. Therefore, detection of SDF is important for fertility testing and assisted reproductive technology.

[0003] Traditional methods for detecting SDF include sperm chromatin structure assay (SCSA), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay, DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) assay, comet assay (CA) and sperm chromatin dispersion (SCD) assay.

[0004] Comet analysis (CA), also known as single-cell gel electrophoresis (SCGE), is a sensitive technique for detecting SDF (sperm-depleted DNA). The CA procedure involves embedding sperm cells in agarose gel mounted on a microscope slide. The slide is then immersed in a lysis solution to disrupt and open the cell membrane, lysing proteins within the cell (e.g., protamine). The agarose gel is then exposed to an electric field, causing negatively charged DNA fragments to migrate towards the anode, creating a comet-like structure. In this comet-like structure, the undamaged DNA nucleoid represents the head, while the damaged DNA tail represents the tail. After staining the DNA with a fluorescent dye, the comet-like structure can be observed using a fluorescence microscope. Analysis of the comet tail can be performed manually or using software; the fluorescence intensity of the comet tail represents the degree of DNA damage. However, due to processes such as electrophoresis and software analysis, CA is complex and time-consuming to perform, making it difficult for CA to meet industry needs.

[0005] The SCD assay is a modified halo assay that uses chemical methods to detect SDF (sperm free radical). The procedure involves embedding sperm cells in an agarose gel, followed by DNA denaturation and deproteinization. Specifically, during DNA denaturation, the double-stranded (DS) DNA in each sperm cell is denatured into single-stranded (SS) DNA. During deproteinization, nuclear proteins (including protamine) in each sperm cell are cleaved, thus dispersing DNA loops from the nuclear proteins to the periphery of each sperm cell. After staining the DNA with 4',6-diamidino-2-phenylindole (DAPI) or Diff-Quik reagent, the dispersed DNA loops can be monitored using a fluorescence microscope or a bright-field optical microscope. Compared to sperm cells without DNA fragmentation, sperm cells with DNA fragmentation have smaller DNA loops that are more difficult to stain. More specifically, sperm cells without DNA fragmentation exhibit a large halo at the head (i.e., the width of the halo is at least one-third of the sperm cell head diameter). Conversely, sperm cells with DNA fragmentation exhibit a small halo or no halo at the head (i.e., the width of the halo is at least less than one-third of the sperm cell head diameter). However, determining the width of the halo is difficult.

[0006] Rapid and accurate methods for SDF determination are reported in U.S. Patent No. 11,644,455 and U.S. Patent Publication No. 2022 / 0195492; these are also included in this case.

[0007] Gram staining is the most widely used and complex staining procedure in bacteriology. Through a series of staining and destaining steps, organisms in the domain of bacteria are classified based on the composition of their cell walls. Gram-positive bacteria have cell walls containing a thick layer of peptidoglycan (comprising 90% of the cell wall); they stain purple. Gram-negative bacteria have cell walls containing a thin layer of peptidoglycan (comprising 10% of the cell wall) and a high lipid content; they stain pink. Gram staining of any sample requires four basic steps: applying the primary stain (crystal violet) to a heat-fixed smear, adding Gram's iodine as a mordant, rapidly destaining with ethanol, acetone, or a combination of ethanol and acetone, and finally counterstaining with safranin.

[0008] Hematoxylin and eosin stain (H&E stain) is commonly used in various areas of histology laboratories, including frozen sections, fine-needle aspiration (FNA), and paraffin-fixed embedded tissue. The H&E staining procedure follows a basic process: dewaxing, dehydration, hematoxylin staining, differentiation, bluing, eosin staining, dehydration, washing, and covering with a slide. Hematoxylin is used to reveal nuclear details within cells. The staining depth depends not only on the amount of DNA in the cell nucleus but also on the length of time the sample is immersed in hematoxylin. Eosin is used as a counterstain to distinguish the cell nucleus from the cytoplasm. It is typically pink, with different shades of pink representing different types of connective tissue fibers. Attached Figure Description

[0009] Figures 1A-1F An embodiment of the method is shown for detecting sperm double-stranded DNA fragmentation (SDF) in a semen sample.

[0010] Figures 2A-2F An embodiment of the method is shown for detecting sperm single-stranded DNA fragmentation (SDF) in a semen sample. Detailed Implementation

[0011] definition As used herein, a "slide" refers to a solid substrate on which a sample, reagent, solution, or other optional substance (such as a gel) can be added. A slide provides a platform (a substrate) upon which biochemical materials are added and reacted. Slides can be made of any solid material, such as glass, plastic, metal, ceramic, or quartz. Glass or plastic are preferred materials.

[0012] As used herein, a "cover glass slide" refers to a solid material that is typically smaller and thinner than the glass slide. A cover glass slide is a material placed on top of the glass slide to cover it. Cover glass slides can be made of any solid material, such as glass, plastic, metal, ceramic, or quartz. Glass or plastic are preferred materials. In one embodiment, the cover glass slide is transparent.

[0013] This invention provides an improved method for performing an experiment on a glass slide, wherein the final reaction product is stained using a coverslip coated with a dye. The method uses a coverslip coated on one side with one or more dyes to stain the product after a chemical reaction has occurred on a glass slide. The coverslip is placed on top of the glass slide with the coated side facing down, thereby staining the product after the reaction has completed on the slide. This invention simplifies and accelerates the staining process for reactions completed on a glass slide.

[0014] The reaction products of staining and embedding sample cells

[0015] A first aspect of the present invention is a method for staining a reaction product, comprising: embedding a sample cell on a glass slide, performing a chemical reaction on the glass slide, staining the reaction product with a coverslip coated with a dye, and removing the colored slide.

[0016] The method comprises the following steps in sequence: (a) embedding a sample cell onto a glass slide; (b) performing one or more chemical reactions on the sample cell embedded in the glass slide to generate a reaction product on the glass slide; (c) contacting the reaction product with a coated side of a coverslip for a period of time, wherein the coated side of the coverslip is coated with one or more dyes to stain the reaction product with the dyes; (d) removing the coverslip from the stained reaction product without damaging the reaction product; and (e) obtaining the stained reaction product on the glass slide.

[0017] In one embodiment, the method further comprises a step (b1) after step (b) and before step (c): removing excess liquid from the slide to make the slide free of liquid flow, and subsequently applying an aqueous solution to wet the reaction product.

[0018] In one embodiment, the method further includes a step (d1) after step (d) and before step (e): rinsing the stained reaction product to remove unbound dye, and subsequently drying the stained product.

[0019] In one embodiment, the method further includes a step (f) after step (e): placing the stained reaction product on the slide under a microscope to examine the stained reaction product.

[0020] In one embodiment, the sample cells are embedded in a gel containing acrylamide, acrylic acid, methacrylic acid, N-isopropylacrylamide (NIPAM), agarose, alginate, polyethylene glycol (PEG), or vinyl chloride.

[0021] In one embodiment, the one or more dyes are selected from the group consisting of: Wright-Giemsa solution, Diff-Quik dye, propidium iodide (PI), SYBR Green, 4',6-dimethylamidine-2-phenylindole (DAPI) dye, and acridine orange (AO).

[0022] In one embodiment, the sample cells are sperm cells from a semen sample.

[0023] In one embodiment, the method is a cell chromatin dispersion assay, which is used to detect sperm DNA fragmentation (SDF) in a semen sample by using a coverslip, wherein one or more staining agents for staining DNA are coated on one side of the coverslip. The coverslip is placed on the slide after a chemical reaction has been carried out on the slide to stain the DNA.

[0024] For SDF detection, the chemical reaction in step (b) may include treating the gel containing sperm cells with a DNA denaturing solution to denature the DNA in the sperm cells.

[0025] For SDF detection, the chemical reaction in step (b) may include treating the gel containing sperm cells with a lysis solution to lyse the nucleoproteins in the sperm cells embedded in the gel. In one embodiment, the lysis solution contains 0.5-4M urea and 0.05-0.5 w / v% SDS.

[0026] SDF detection method

[0027] In one embodiment, this method detects sperm DNA fragmentation (SDF) in a semen sample. The method sequentially comprises the following steps: (a) embedding the semen sample containing sperm cells in a gel on a glass slide to obtain a sperm cell-embedded gel fixed on the glass slide; (b) treating the sperm cell-embedded gel with a lysis solution to lyse the nucleoproteins in the sperm cells embedded in the gel; (c) removing the liquid from the gel; (d) providing a coverslip, wherein one side of the coverslip is coated with one or more DNA stains; (e) using an aqueous solution (e.g., P... (c) Apply an aqueous solution (BS or an ethanol-water solution) to the gel in step (c) to moisten the gel; (f) Contact the coated side of the coverslip with the moistened gel to stain the DNA; (g) Remove the coverslip from the stained gel; (h) Rinse the stained gel to remove unbound dye; (i) Dry the rinsed gel; and (j) Examine the dried gel to observe whether a halo forms around the head of each sperm cell to determine SDF.

[0028] In one embodiment, the semen sample is a human semen sample.

[0029] In one embodiment, the semen sample is diluted with a diluent to achieve a sperm cell concentration of 4 × 10⁻⁶. 6 Cells / mL to 2.8 × 10⁻⁶ 7 Cells / mL. Examples of diluents may include, but are not limited to, Earle's medium, human tubal fluid (HTF) medium, tris-buffered saline (TBS), phosphate-buffered saline (PBS), or physiological saline.

[0030] DNA staining agents suitable for this method include, but are not limited to, Wright-Giemsa solution, Diff-Quik stain (0.4% Asure B plus 0.125% Eosin Y), propidium iodide (PI), SYBR Green, 4',6-dimethylamidine-2-phenylindole (DAPI) stain, and acridine orange.

[0031] Example 1: SDF Detection

[0032] In Example 1, this method detects sperm double-stranded DNA fragmentation (SDF) in a semen sample. After the reaction is complete, the dried gel is examined to observe whether an aura forms around the head of the sperm cells; the presence of an aura indicates the presence of SDF.

[0033] In one embodiment, step (a) comprises: embedding the semen sample containing sperm cells in a gel containing acrylamide, acrylic acid, methacrylic acid, N-isopropylacrylamide (NIPAM), alginate, or polyethylene glycol (PEG) to obtain a gel containing sperm cells. In a preferred embodiment, the gel is a polyacrylamide gel.

[0034] In one embodiment, step (b) comprises treating the sperm-embedded gel with a lysis solution comprising 0.5 M to 4 M urea and 0.05% (w / v, g / mL) to 0.5% (w / v, g / mL) sodium dodecyl sulfate (SDS), thereby lysing the nucleoproteins in the sperm cells embedded in the gel. For example, the lysis solution comprises 0.5-4 M urea and 0.05-0.5% w / v SDS.

[0035] In one embodiment, sodium dodecyl sulfate in the lysis solution is used as an ionic surfactant, and urea is used as a protein denaturant. These two components enhance the lysis of protamine, thus allowing DNA loops to be readily released from the protamine to the periphery of the sperm cell head, where they are then monitored by forming an aura with a DNA stain, thereby reducing the lysis time (e.g., less than 5 minutes). The lysis solution may further contain an additional ionic or nonionic surfactant.

[0036] For example, the additional ionic surfactant may be selected from the group consisting of sodium deoxycholate, sodium cholate, sodium lauroylsarcosinate, and combinations thereof. In a particular embodiment, the additional nonionic surfactant may be selected from the group consisting of Triton X-100, Nonoxynol-40 (NP-40), Pluronic F-127 (F-127), Tween-20, and combinations thereof. In an exemplary embodiment, the additional nonionic surfactant is Triton X-100.

[0037] In one embodiment, the lysis solution may further contain an additional protein denaturant. Examples of the additional protein denaturant may include, but are not limited to, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate, guanidinium chloride, or a combination thereof.

[0038] In one embodiment, the lysis solution may further comprise a reducing agent. Examples of the reducing agent may include, but are not limited to, dithiothreitol (DTT), β-mercaptoethanol, dithioerythritol (DTE), tributylphosphine (TBP), tris(2-carboxyethyl)phosphine (TCEP), hydrochloride, or combinations thereof. In an exemplary embodiment, the reducing agent is TCEP.

[0039] In one embodiment, the lysis solution may further contain salts. Examples of salts may include, but are not limited to, sodium chloride (NaCl), potassium chloride (KCl), or combinations thereof.

[0040] In one embodiment, the lysis solution may further comprise a titrant. Examples of the titrant may include, but are not limited to, sodium hydroxide (NaOH), hydrochloric acid (HCl), or combinations thereof.

[0041] In one embodiment, the lysis solution may further comprise 0.15 M to 3 M NaCl, 0.05 M to 0.2 M DTT, 0.1% (v / v) to 5% (v / v) Triton X-100, and 0.01 M to 0.02 M NaOH. In an exemplary embodiment, the lysis solution comprises 1 M urea, 0.05% (w / v) SDS, 2.5 M NaCl, 0.1 M DTT, 1% (v / v) Triton X-100, and 0.02 M NaOH. In another exemplary embodiment, the lysis solution comprises 4 M urea, 0.05% (w / v) SDS, 0.15 M NaCl, 0.2 M DTT, 0.5% (v / v) Triton X-100, and 0.01 M NaOH. In yet another exemplary embodiment, the lysis solution comprises 0.5 M urea, 0.5% (w / v) SDS, 3 M NaCl, 0.05 M DTT, 5% (v / v) Triton X-100 and 0.015 M NaOH.

[0042] The lysis solution can be adjusted to have a desired pH value. In one particular embodiment, the lysis solution may have a pH value in the range of 7 to 9. In an exemplary embodiment, the lysis solution may have a pH value in the range of 7 to 8.2. In another exemplary embodiment, the lysis solution has a pH value of 7.5.

[0043] In one embodiment, step (a) of the method comprises embedding the semen sample containing sperm cells in a polyacrylamide gel containing 3-22% (w / v) acrylamide to obtain a polyacrylamide gel embedded with sperm cells; and step (b) of the method comprises subjecting the polyacrylamide gel embedded with sperm cells to a lysis reaction in a lysis solution containing 0.5 M to 4 M urea and 0.05-0.5% (w / v) SDS.

[0044] In a specific embodiment, in step (a), the acrylamide concentration in the polyacrylamide gel is 3-22% (w / v), 3-16% (w / v), or 4-16% (w / v). In one embodiment, the polyacrylamide gel is formed from acrylamide and bis-acrylamide, and the ratio of acrylamide to bis-acrylamide is from 19:1 (w / w) to 37.5:1 (w / w).

[0045] In a particular embodiment, in step (a), the pore size in the polyacrylamide gel may be from 3 nanometers (nm) to 10 nm or from 3 nm to 9 nm.

[0046] After the gel is moistened with an aqueous solution (e.g., phosphate-buffered saline, PBS), in step (e), while the gel is still moist, the moistened gel is immediately brought into contact with the coated side of the coverslip to stain the DNA. For example, the contact time between the moistened gel and the stain is less than 1 minute, less than 2 minutes, less than 5 minutes, less than 10 minutes, or less than 30 minutes.

[0047] Figures 1A-1F An embodiment of a first aspect method for detecting sperm double-stranded DNA fragmentation (SDF) in a semen sample is shown.

[0048] Example 2: SDF Detection

[0049] In Example 2, this method detects sperm DNA fragmentation (SDF) in a semen sample, which involves denaturing sperm DNA into single-stranded DNA.

[0050] The method comprises the following steps in sequence: (a) embedding a semen sample containing sperm cells in a gel to obtain a sperm cell-embedded gel; (aa) treating the sperm cell-embedded gel with a DNA denaturing solution to denature the DNA in the sperm cells; (b) treating the sperm cell-embedded gel with a lysis solution to lyse the nucleoproteins in the sperm cells embedded in the gel; (c) removing the liquid from the gel; and (d) obtaining a coverslip, wherein one side of the coverslip is coated with one or more... (e) Applying an aqueous solution to the gel from step (c) to moisten the gel; (f) Contacting the moistened gel with the coated side of a coverslip to stain the DNA; (g) Removing the coverslip from the stained gel; (h) Rinsing the stained gel to remove unbound dye; (i) Drying the rinsed gel; and (j) Examining the dried gel to observe whether a halo forms around the head of each sperm cell to determine SDF.

[0051] In one embodiment, the gel comprises agarose, acrylamide, alginate, or vinyl chloride.

[0052] In one embodiment, the gel may be an agarose gel, and the concentration of agarose in the agarose gel may be 1-3% (w / v). In an exemplary embodiment, the concentration of agarose in the agarose gel is 1.25% (w / v).

[0053] In one embodiment, the lysis solution comprises urea at a concentration of 0.5 M to 4 M and sodium dodecyl sulfate at a concentration of 0.05% (w / v) to 0.5% (w / v) to lyse nucleoproteins in sperm cells.

[0054] In one embodiment, the agarose gel may be melted at a temperature of 95°C to 100°C using a microwave oven or a constant-temperature water bath before being used to embed the semen sample. In an exemplary embodiment, the agarose gel is melted at a temperature of 95°C.

[0055] In one embodiment, the DNA denaturing solution may comprise an acidic aqueous solution or an alkaline aqueous solution and have an equivalent concentration of 0.05 N to 0.08 N. In a particular embodiment, the DNA denaturing solution has an equivalent concentration of 0.06 N to 0.07 N.

[0056] In one embodiment, the DNA denaturing solution may be an acidic aqueous solution comprising an acid selected from the group consisting of hydrochloric acid, acetic acid, nitric acid, sulfuric acid, and combinations thereof. In a particular embodiment, the DNA denaturing solution is an acidic aqueous solution containing hydrochloric acid.

[0057] In one embodiment, the DNA denaturing solution may be an alkaline aqueous solution comprising a base selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, and combinations thereof. In a particular embodiment, the DNA denaturing solution is an alkaline aqueous solution containing sodium hydroxide.

[0058] In the lysis solution, sodium lauryl sulfate (SDS), also known as sodium dodecyl sulfate, is used as an ionic surfactant, and urea is used as a protein denaturant. These two components enhance the lysis of protamine, thus allowing DNA loops to be easily released from the protamine to the periphery of the sperm cell head, where they are then detected by forming a halo effect with a DNA stain. The use of SDS and urea in the lysis solution effectively reduces the lysis time (e.g., less than 5 minutes), thereby reducing sample uncertainty. According to the invention, the lysis solution may further contain an additional ionic or nonionic surfactant.

[0059] In a particular embodiment, the lysis solution may contain an ionic surfactant selected from the group consisting of sodium deoxycholate, sodium cholate, sodium lauroyl sarcosinate, and combinations thereof.

[0060] In a particular embodiment, the lysis solution may contain a nonionic surfactant selected from the group consisting of Triton X-100, NP-40, F-127, Tween-20, and combinations thereof. In an exemplary embodiment, the nonionic surfactant is Triton X-100.

[0061] In certain embodiments, the lysis solution may further contain an additional protein denaturant. Examples of the additional protein denaturant may include, but are not limited to, 3-[3-(cholanamidopropyl)dimethylammonium]propanesulfonic acid hydrate, guanidine hydrochloride, or combinations thereof.

[0062] In a particular embodiment, the lysis solution may further comprise a reducing agent. Examples of the reducing agent may include, but are not limited to, dithiothreitol (DTT), tris(carboxyethyl)phosphine (TCEP), dithioerythritol (DTE), β-mercaptoethanol (β-ME), glutathione (GSH), dimercaprol, heparin, or combinations thereof. In an exemplary embodiment, the reducing agent is DTT or TCEP.

[0063] In certain embodiments, the lysis solution may further comprise salts. Examples of such salts may include, but are not limited to, sodium chloride (NaCl), potassium chloride (KCl), or combinations thereof.

[0064] In a particular embodiment, the lysis solution may further comprise a pH titrant. Examples of the titrant may include, but are not limited to, sodium hydroxide (NaOH), hydrochloric acid (HCl), or combinations thereof.

[0065] In a particular embodiment, the lysis solution may further comprise 0.15 M to 3 M NaCl, 0.05 M to 0.2 M DTT or TCEP, 0.1% (v / v) to 5% (v / v) Triton X-100 and 0.01 M to 0.02 M NaOH.

[0066] In one exemplary embodiment, the lysis solution comprises 1 M urea, 0.05% (w / v, g / mL) SDS, 2.5 M NaCl, 0.1 M DTT or 0.05 M TCEP, 1% (v / v) Triton X-100, and 0.02 M NaOH. In another exemplary embodiment, the lysis solution comprises 4 M urea, 0.05% (w / v, g / mL) SDS, 0.15 M NaCl, 0.2 M DTT or 0.05 M TCEP, 0.5% (v / v) Triton X-100, and 0.01 M NaOH. In yet another exemplary embodiment, the lysis solution comprises 0.5 M urea, 0.5% (w / v, g / mL) SDS, 3 M NaCl, 0.05 M DTT or TCEP, 5% (v / v) Triton X-100 and 0.015 M NaOH.

[0067] According to this disclosure, the lysis solution can be adjusted to a desired pH value using the titrant. In a particular embodiment, when the DNA denaturing solution is an acidic aqueous solution, the lysis solution may have a pH value in the range of 7.5 to 9.0. In a particular embodiment, when the DNA denaturing solution is an alkaline aqueous solution, the lysis solution may have a pH value in the range of 5.5 to 7.0. In an exemplary embodiment, the lysis solution has a pH value in the range of 8.2 to 8.5.

[0068] Example 3: SDF Detection

[0069] In Example 3, this method provides another method comprising denaturing sperm DNA into single-stranded DNA.

[0070] The method comprises: (a) heating an agarose solution, followed by adding a DNA denaturing solution and the semen sample containing sperm cells to form a mixture; (b) subjecting the mixture to a gel polymerization reaction to obtain an agarose gel in which the sperm cells containing denatured DNA are embedded; and (c) using a solution containing urea at a concentration of 0.5 M to 4 M and urea at a concentration of 0.05-0.5%. (d) Lysing the agarose gel with a (w / v) sodium dodecyl sulfate lysis solution, thereby lysing the nucleoproteins in the sperm cells embedded in the agarose gel; (e) Removing the liquid from the gel; (f) Obtaining a coverslip, one side of which is coated with one or more DNA stains; (g) Applying an aqueous solution to the gel from step (d) to moisten the gel; (h) Contacting the moistened gel with the coated side of the coverslip to stain the DNA; (i) Removing the coverslip from the stained gel; (j) Rinsing the stained gel to remove unbound stains; (k) Drying the rinsed gel; and (d) Examining the dried gel to observe whether a halo forms around the head of each sperm cell to determine SDF.

[0071] In step (a), prior to heating, the agarose solution may be further mixed with the aforementioned acid-base indicator.

[0072] In step (a), the heat treatment may be performed at a temperature of 95°C to 100°C. In an exemplary embodiment, the heat treatment is performed at a temperature of 95°C.

[0073] Example 4: SDF Detection

[0074] In Example 4, this method provides another method comprising denaturing sperm DNA into single-stranded DNA.

[0075] The method comprises: (a) mixing the semen sample containing sperm cells with a DNA denaturing solution and a gel-forming component, and then subjecting the resulting mixture to a gel polymerization reaction to obtain a gel in which the sperm cells containing denatured DNA are embedded, the gel-forming component being selected from the group consisting of acrylamide, alginate, and vinyl chloride; (b) using a solution containing urea at a concentration of 0.5 M to 4 M and urea at a concentration of 0.05-0.5%. (c) Lyse the gel with a lysis solution of sodium dodecyl sulfate (w / v) to lyse the nucleoproteins embedded in the sperm cells in the gel; (d) Remove the liquid from the gel; (e) Obtain a coverslip, one side of which is coated with one or more DNA stains; (f) Apply an aqueous solution to the gel of step (c) to moisten the gel; (g) Contact the moistened gel with the coated side of the coverslip to stain the DNA with the stain; (h) Remove the coverslip from the stained gel; (i) Rinse the stained gel to remove unbound stains; (i) Dry the rinsed gel; and (ii) Examine the dried gel to observe whether a halo forms around the head of each sperm cell to determine SDF.

[0076] In the methods of Examples 2, 3, and 4, after the gel is moistened with an aqueous solution (e.g., an aqueous ethanol solution), in step (e), while the gel is still moist, the moistened gel is immediately brought into contact with the coated side of the coverslip to stain the DNA. For example, the contact time between the moistened gel and the stain is less than 1 minute, less than 2 minutes, less than 5 minutes, less than 8 minutes, or less than 10 minutes.

[0077] In the methods of Examples 2 to 4, the stained gel was examined to observe whether a halo was generated near the head of the sperm cell; wherein the absence of a halo or the width of the halo being at least one-third of the diameter of the head of the corresponding sperm cell represented the presence of sperm DNA fragmentation.

[0078] In the methods of Examples 3 and 4 above, the general reagents are similar to those in Example 2.

[0079] Figures 2A-2F The methods of Examples 2 to 4 are shown for detecting sperm single-stranded DNA fragmentation (SDF) in semen samples.

[0080] In all of the above methods, after the step of observing whether a halo is formed near the head of sperm cells, the method optionally further includes a step of calculating the DNA fragmentation index (DFI) (%), which represents the percentage of sperm cells with DNA fragmentation in the total number of sperm cells. Generally, a normal semen sample has a DFI ≤ 15%; an abnormal semen sample has a DFI ≥ 30%. Those in between (15% < DFI < 30%) are considered borderline samples or threshold samples.

[0081] Staining and morphological examination

[0082] A second aspect of the present invention is a method for staining and examining the morphology of a reaction product. The method includes adhering a sample on a slide, performing one or more reactions in the sample on the slide, staining the reaction product with a coverslip coated with a stain, and examining the morphology of the sample without removing the coverslip. The method is applicable to examining the morphology of bacteria, sperm cells, and tissues.

[0083] In the present method for staining and examining the morphology of a reaction product, the method sequentially includes the following steps: (a): adhering a sample on a slide, where the sample is selected from the group consisting of tissue, body fluid, microorganism, and cell; (b): performing one or more reactions in the sample on the slide, thereby adhering a reaction product on the slide; (c): contacting the reaction product with a coated side of a coverslip for a period of time, where one or more stains are coated on the coated side of the coverslip to stain the reaction product through the stains; and (d): placing the slide, the stained reaction product, and the coverslip under a microscope to examine the morphology of the stained reaction product.

[0084] In one embodiment, adhering a sample on a slide in step (a) includes fixing a smear sample on a slide.

[0085] In one embodiment, adhering a sample on a slide in step (a) adheres a paraffin section of a tissue sample on a slide through cross-sectional surface interaction.

[0086] In one embodiment, adhering a reaction product on the slide in step (b) includes dehydrating to fix the reaction product on the slide.

[0087] In one embodiment, this method is applied to bacterial staining, such as Gram staining. Gram staining is the most widely used staining procedure in bacteriology. For Gram staining, in step (a), a bacterial smear sample is applied to a glass slide, air-dried, and heat-fixed onto the slide. In step (b), the bacterial cell line is reacted with crystal violet for staining, washed, and then stained with Gram's iodine solution. After washing, it is reacted with an excess of destaining agent (e.g., ethanol, acetone, or a mixture of ethanol and acetone) until the excess destaining agent becomes clear. In step (c), the coated side of a coverslip is contacted with the reaction product from step (b) for a period of time (e.g., 30 seconds to 2 minutes), wherein the coated side of the coverslip is coated with a red counterstain to stain the reaction product. The stained reaction product, the glass slide, and the coverslip are optionally washed and blotted together. In step (d), the slide, the stained reaction product, and the coverslip are placed under a microscope to examine the morphology of the stained reaction product. Gram-negative bacteria are stained pink / red, while Gram-positive bacteria are stained blue / purple.

[0088] In one embodiment, this method is applied to sperm cell morphology staining, such as Diff-quick staining, Papanicolaou staining, Shorter staining, HE staining, Wright staining, and Wright-Giemsa staining, and measures parameters of the sperm cell head and evaluates the staining effect of the sperm.

[0089] For example, in the Diff rapid staining method, in step (a), a dried sperm smear from a semen sample is applied to a glass slide and fixed. In step (b), the smear is reacted with a solution containing methanol and eosin and then washed. In step (c), the coated side of a coverslip is contacted with the reaction product from step (b) for a period of time (e.g., 30 seconds to 2 minutes), wherein the coated side of the coverslip is coated with methylene blue to stain the reaction product. The stained reaction product, the glass slide, and the coverslip are washed and dried together. In step (d), the glass slide, the stained reaction product, and the coverslip are placed under a microscope to examine the morphology of the stained reaction product (the maximum length and width of the sperm cell head).

[0090] For example, in Burley's staining, in step (a), a sperm smear from a semen sample is applied to and fixed onto a glass slide. The smear is optionally rinsed with an aqueous ethanol solution and water. In step (b), the smear is reacted with hematoxylin staining solution (Harris) and then washed. Subsequently, the smear is immersed in one or more aqueous ethanol solutions for staining with orange G staining solution, and then immersed in an aqueous ethanol solution. In step (c), a coverslip with its coated side in contact with the reaction product from step (b) for a period of time (e.g., 30 seconds to 2 minutes), wherein the coated side of the coverslip is coated with EA36 staining solution (brightgreen and eosin) to stain the reaction product. The stained reaction product, the glass slide, and the coverslip are optionally immersed together in ethanol and then blotted dry. In step (d), the slide, the stained reaction product, and the coverslip are placed under a microscope to examine the morphology of the stained reaction product (the maximum length and width of the sperm cell head).

[0091] For example, in the Shor staining method, in step (a), a dried sperm smear from a semen sample is applied to a glass slide and fixed to the slide in a solution containing methanol and triarylmethane, and then washed. In step (b), the smear is reacted with hematoxylin staining solution (Harris) and washed. Subsequently, the smear is immersed in an acidic aqueous ethanol solution and washed, and then immersed again in an aqueous ethanol solution. In step (c), the coated side of a coverslip is contacted with the reaction product from step (b) for a period of time (e.g., 30 seconds to 2 minutes), wherein the coated side of the coverslip is coated with Shor staining solution to stain the reaction product. The stained reaction product, the glass slide, and the coverslip may optionally be immersed together in ethanol and blotted dry. In step (d), the slide, the stained reaction product, and the coverslip are placed under a microscope to examine the morphology of the stained reaction product (the maximum length and width of the sperm cell head).

[0092] In one embodiment, this method is applied to hematoxylin-eosin (H&E) staining of a tissue sample. H&E is a combination of two histological stains: hematoxylin and eosin. Hematoxylin stains the cell nucleus purple-blue, while eosin stains the extracellular matrix and cytoplasm pink, with other structures exhibiting varying shades, tones, and combinations of these colors.

[0093] In the hematoxylin-eosin method, in step (a), a paraffin section of a tissue is applied to and adhered to a glass slide. In step (b), the paraffin section is dewaxed and rehydrated with xylene, ethanol, or water; then reacted with hematoxylin stain and washed and blotted dry. In step (c), a coverslip with its coated side in contact with the reaction product from step (b) for a period of time (e.g., 30 seconds to 2 minutes), wherein the coated side of the coverslip is coated with eosin to stain the reaction product by eosin. The stained reaction product, the glass slide, and the coverslip are washed together and optionally dehydrated. In step (d), the glass slide, the stained reaction product, and the coverslip are placed under a microscope to examine the morphology of the cell nuclei, cytoplasm, and extracellular matrix in the stained reaction product.

[0094] It should be understood that the foregoing description describes preferred embodiments of the present invention, and modifications may be made thereto without departing from the scope of the invention as set forth in the claims.

Claims

1. A method for staining a reaction product, comprising the following steps in sequence: (a): A sample of cells is embedded on a glass slide; (b): One or more chemical reactions are carried out in sample cells embedded in the slide to produce a reaction product on the slide; (c): The reaction product is contacted with the coated side of a coverslip for a period of time, wherein one or more dyes are coated on the coated side of the coverslip to stain the reaction product by the dyes. (d): Remove the coverslip from the stained reaction product without damaging the stained reaction product; and (e): The stained reaction product is obtained on the slide.

2. The method of claim 1, further comprising a step (b1) after step (b) and before step (c): (b1) Remove excess liquid from the slide to make the slide free of liquid flow, and then apply an aqueous solution to wet the reaction product.

3. The method of claim 1, further comprising a step (d1) after step (d) and before step (e): (d1) Rinse the stained reaction product to remove unbound dye and dry the stained product.

4. The method of claim 1, further comprising a step (f) after step (e): (f) The stained reaction product on the slide is placed under a microscope to examine the stained reaction product.

5. The method of claim 1, wherein, The sample cells were embedded in a gel containing acrylamide, acrylic acid, methacrylic acid, N-isopropylacrylamide, agarose, alginate, polyethylene glycol, or vinyl chloride.

6. The method according to any one of claims 1 to 5, wherein, The one or more dyes are selected from the group consisting of: Wright-Gymsa solution, Diff-Quik dye, propidium iodide, SYBR Green, 4',6-dimethylamino-2-phenylindole dye, and acridine orange.

7. The method according to any one of claims 1 to 6, wherein the method is a cell chromatin dispersion assay.

8. The method of claim 7, wherein, The sample cells were sperm cells from a semen sample.

9. The method of claim 8, wherein, The chemical reaction in step (b) involves treating the gel containing sperm cells with a DNA denaturing solution to denature the DNA in the sperm cells.

10. The method of claim 1 or 9, wherein, The chemical reaction in step (b) involves treating the gel containing sperm cells with a lysis solution to lyse the nucleoproteins in the sperm cells embedded in the gel.

11. The method of claim 10, wherein, The lysis solution contains 0.5-4 M urea and 0.05-0.5% w / v SDS.

12. A method for detecting sperm DNA fragmentation in a semen sample, comprising the following steps in sequence: (a): On a glass slide, the semen sample containing sperm cells is embedded in a gel to obtain a gel containing sperm cells fixed on the glass slide; (b): The gel containing sperm cells is treated with a lysis solution to cause the nucleoproteins in the sperm cells embedded in the gel to lyse. (c): Remove the liquid from the gel; (d): Obtain a coverslip, wherein one side of the coverslip is coated with one or more DNA dyes; (e): Apply an aqueous solution (e.g., PBS or an aqueous ethanol solution) to the gel in step (c) to wet the gel; (f): The coated side of the coverslip is brought into contact with the moistened gel to stain the DNA through the dye. (g): Remove the coverslip from the stained gel; (h): Rinse the stained gel to remove unbound dye; (i): Dry the rinsed gel; as well as (j): Examine the dried gel to observe whether a halo forms around the head of each sperm cell to determine SDF.

13. A method for staining and examining the morphology of a reaction product, comprising the following steps in sequence: (a): A sample is attached to a glass slide, wherein the sample is selected from a group consisting of tissues, body fluids, microorganisms and cells; (b): One or more reactions are carried out on the sample on the slide to obtain a reaction product that adheres to the slide; (c): The reaction product is contacted for a period of time with the coated side of a coverslip, wherein one or more dyes are coated on the coated side of the coverslip to stain the reaction product with the dyes; and (d): The slide, the stained reaction product and the coverslip are placed under a microscope to examine the morphology of the stained reaction product.

14. The method of claim 13, wherein, This method is used for bacterial staining, and the dye is safranin.

15. The method of claim 13, wherein, This method is used for staining sperm cell morphology, and the staining agent is methylene blue, EA36, or Schauer's staining solution.

16. The method of claim 13, wherein, This method is used for staining tissue samples, and the staining agent is eosin.

17. The method according to any one of claims 1 to 16, wherein, The cover glass is made of glass or plastic.

Images