A ferrous ion fluorescent staining detection kit and method

Abstract

The application relates to the field of biomedical detection technology and discloses a ferrous ion fluorescent staining detection kit and method.The application realizes in-situ spatial positioning detection of ferrous ions in paraffin tissue sections through dewaxing repair, glycine quenching, ascorbic acid reduction sensitization and probe-immune double staining, overcomes the defects that the existing fluorescent probe method is only suitable for living cells; ascorbic acid reduces the oxidized Fe 3+ in fixing to Fe 2+ , significantly improves the detection sensitivity; the double immunofluorescence co-localization of ferrous ions and GPX4 or ACSL4 on the section establishes a quantitative evaluation standard of the ferrous ion content and the degree of ferroptosis; the glycine quenching reduces the spontaneous fluorescence background, guarantees a high signal-to-noise ratio; the method is suitable for paraffin sections, frozen sections and cell samples and has wide clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a ferrous ion fluorescent staining detection kit and method. Background Technology

[0002] Ferroptosis is a programmed cell death mechanism dependent on lipid peroxidation caused by intracellular iron accumulation. Its essence lies in the imbalance of the intracellular antioxidant system, specifically due to the accumulation of intracellular ferrous ions (Fe2+). 2+ Abnormal increases in ferrous ions lead to the Fenton reaction with H2O2, generating hydroxyl radicals that promote lipid peroxidation of polyunsaturated fatty acids, ultimately resulting in cell membrane damage and mitochondrial dysfunction. Ferroplasm is closely related to the physiological processes of various diseases such as tumors, Parkinson's disease, and liver and kidney damage. Among the indicators for detecting ferroplasm, the detection of ferrous ion content is one of the key markers for determining the occurrence of ferroplasm. Currently, the main methods for detecting ferrous ions include the following: (1) Live cell imaging based on fluorescent probes, such as using fluorescent probes like RhoNox-6 and FerroOrange to detect ferrous ions in live cells. 2+ A specific reaction occurs, and the change in fluorescence intensity is detected by fluorescence microscopy or flow cytometry to achieve real-time dynamic monitoring of ferrous ions in living cells; (2) a colorimetric reagent kit detection method, such as using chromogenic reagents such as Ferrozine or TPTZ to react with Fe 2+ A colored complex is formed, and the absorbance value is measured by a spectrophotometer for quantitative analysis. It is suitable for liquid samples such as cell lysate, serum or tissue homogenate; (3) The total iron quantification method based on inductively coupled plasma mass spectrometry (ICP-MS) is to ionize the iron element in the sample by high-temperature plasma and then perform mass spectrometry analysis. It can accurately determine the total iron content in the sample, but it cannot distinguish Fe 2+ with Fe 3+ .

[0003] However, the aforementioned existing technologies have the following technical limitations in detecting ferrous ions in ferroptosis-related pathological tissues: First, existing fluorescent probe methods (such as RhoNox-6) are mainly suitable for detecting live cells or fresh samples. For clinically commonly used formaldehyde-fixed or paraffin-embedded tissue sections, due to the presence of ferrous ions during fixation... 2+ Easily oxidized to Fe 3+Furthermore, the probes have difficulty penetrating the dense tissue matrix, resulting in a significant decrease in detection sensitivity or even failure to detect, which cannot meet the retrospective testing requirements of archived tissue samples in pathological diagnosis. Second, existing colorimetric and ICP-MS methods require tissue homogenization, which destroys the spatial distribution information of ferrous ions in the tissue, making it impossible to achieve in-situ localization detection of ferrous ions in tissue microregions (such as specific cell types or subcellular structures), and even more impossible to perform spatial co-localization analysis of ferrous ion accumulation and ferroptosis markers (such as GPX4 and ACSL4). Third, existing detection methods are all single-index detections, and cannot simultaneously achieve dual visualization of ferrous ion localization and ferroptosis-related protein expression on the same tissue section, resulting in the inability to establish a direct spatial correspondence between ferrous ion content and the degree of ferroptosis, thus limiting the accuracy of ferroptosis mechanism research and clinical pathological diagnosis.

[0004] Therefore, there is an urgent need to develop a kit and method suitable for fixed tissue section samples, capable of in-situ fluorescence staining detection of ferrous ions, and capable of dual immunofluorescence co-localization analysis with ferroptosis markers, in order to solve the technical problems in the existing technology that cannot detect the spatial localization of ferrous ions in archived tissue samples, and cannot perform co-localization analysis of ferrous ion accumulation with ferroptosis markers. Summary of the Invention

[0005] The purpose of this invention is to provide a ferrous ion fluorescence staining detection kit and method, which solves the problems in the prior art that it is impossible to perform in-situ spatial localization detection of ferrous ions in formaldehyde-fixed and paraffin-embedded tissue section samples, and that it is impossible to perform dual immunofluorescence co-localization analysis of ferrous ion accumulation and ferroptosis markers on the same tissue section.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a method for detecting ferrous ions by fluorescent staining, comprising the following steps: The paraffin embedding agent was removed by heating and incubating with dewaxing solution. After gradient ethanol hydration and washing with distilled water, the tissue sections were microwaved with citrate buffer and naturally cooled to room temperature to obtain tissue sections with fully exposed ferrous ion binding sites. The autofluorescence background generated by aldehyde fixation in the sample was extinguished by incubation with glycine solution at room temperature. After washing with PBS, pre-treated sections with reduced background signal were obtained. By incubating the tissue with a staining working solution containing a fluorescent probe, ascorbic acid reducing agent, HEPES buffer and surfactant in the dark, the probe specifically binds to ferrous ions in the tissue. After washing with PBS, tissue sections with ferrous ion fluorescent signals are obtained. After blocking with goat serum, the primary antibody was added and incubated overnight, followed by incubation with fluorescent secondary antibody in the dark. After washing with PBS, a double-stained section with both ferrous ion fluorescence signal and ferroptosis marker fluorescence signal was obtained. Cell nuclei were counterstained by incubation with DAPI staining solution in the dark, washed with PBS, and mounted with anti-fluorescence quenching mounting medium to obtain complete stained sections that can be preserved for a long time. Ferrous ion, ferroptosis markers, and nuclear fluorescence signals were acquired from sections using a multi-channel laser confocal microscope. The images were then processed by image analysis software to obtain quantitative values ​​of ferrous ion fluorescence intensity and colocalization coefficients. The quantitative values ​​and colocation coefficients were compared with the normal control group to obtain the results of determining whether ferroptosis was positive, suspicious, or negative.

[0007] The section on "removing paraffin embedding agent by heating and incubating with dewaxing solution, followed by gradient ethanol hydration and washing with distilled water, then microwave repair with citrate buffer, and natural cooling to room temperature to obtain tissue sections with fully exposed ferrous ion binding sites" includes the following steps: Place the paraffin-embedded tissue sections in dewaxing solution and incubate at 55-60℃ for 15-30 minutes to remove the paraffin embedding agent; The dewaxed sections were sequentially treated with 100% ethanol, 95% ethanol, 70% ethanol and distilled water for 5 minutes each for gradient hydration. After gradient hydration, the sections were microwaved at 95-100℃ for 15-20 minutes with citrate buffer at pH 6.0. After remedy, the sections were allowed to cool naturally to room temperature to obtain tissue sections with fully exposed ferrous ion binding sites.

[0008] The section on "inhibiting the autofluorescence background generated by aldehyde fixation in the sample by incubation with glycine solution at room temperature, followed by washing with PBS to obtain pre-treated sections with reduced background signal" includes the following steps: Tissue sections were incubated in 0.1-0.3M glycine solution at room temperature for 20-30 minutes to quench the autofluorescence background generated by aldehyde fixatives. The quenched sections were washed three times with PBS buffer for 5 minutes each time to obtain pretreated sections with reduced background signal.

[0009] The step described in "incubating the tissue with a staining working solution containing a fluorescent probe, ascorbic acid reducing agent, HEPES buffer, and surfactant in the dark to allow the probe to specifically bind to ferrous ions in the tissue, followed by washing with PBS to obtain tissue sections with ferrous ion fluorescent signals" includes the following steps: The pretreated sections were placed in a staining working solution containing a ferrous ion-specific fluorescent probe, ascorbic acid reducing agent, HEPES buffer (pH 7.2-7.6), and 0.05%-0.1% Triton X-100, and incubated at 37°C in the dark for 30-60 minutes to allow the probe to specifically bind to ferrous ions in the tissue and generate a fluorescent signal. The stained sections were washed three times with PBS buffer for 5 minutes each time to obtain tissue sections with ferrous ion fluorescence signals.

[0010] The procedure described in the section "After blocking with goat serum, the primary antibody was added sequentially and incubated overnight, followed by incubation with fluorescent secondary antibody in the dark, and then washed with PBS to obtain a double-stained section containing both ferrous ion fluorescence and ferroptosis marker fluorescence signals" includes the following steps: After blocking the sections with 5%-10% normal goat serum at room temperature for 30 minutes, add GPX4 or ACSL4 primary antibody working solution and incubate overnight at 4°C. Wash with PBS 3 times, 5 minutes each time. After incubation with primary antibody, the slides were added to fluorescently labeled secondary antibody working solution and incubated at 37°C in the dark for 45-60 minutes. The slides were then washed three times with PBS for 5 minutes each time to obtain double-stained slides with both ferrous ion fluorescence signals and ferroptosis marker fluorescence signals.

[0011] The section on "counter-staining cell nuclei by incubating with DAPI staining solution in the dark, washing with PBS, and mounting with anti-fluorescence quenching mounting medium to obtain complete stained sections that can be preserved for a long time" includes the following steps: Double-stained sections were incubated with 1 μg / mLDAPI staining solution at room temperature in the dark for 5-10 minutes for nuclear counterstaining, followed by washing twice with PBS for 3 minutes each time. After counterstaining, add anti-fluorescence quenching mounting medium to the slide, cover with a coverslip and seal the edges, and store at 4°C in the dark to obtain a complete stained slide that can be preserved for a long time.

[0012] In a second aspect, a ferrous ion fluorescent staining detection kit is used in the ferrous ion fluorescent staining detection method described in the first aspect, comprising a ferrous ion fluorescent probe, tissue processing solution, blocking solution, washing solution, antibody diluent, nuclear staining solution, and mounting medium.

[0013] This invention provides a fluorescent staining method for detecting ferrous ions. Through steps such as dewaxing and repair, glycine quenching, ascorbic acid reduction and sensitization, probe-immunoassay dual staining, and multi-channel co-localization analysis, this invention achieves in-situ spatial localization detection of ferrous ions in paraffin-embedded tissue sections. This overcomes the limitations of existing fluorescent probe methods, which are only applicable to live cells and cannot detect archived tissue samples. The method utilizes ascorbic acid to remove ferrous ions oxidized during fixation. 3+ In-situ reduction to Fe 2+This significantly improves the detection sensitivity of fixed tissue samples. By achieving dual immunofluorescence co-localization visualization of ferrous ions and ferroptosis markers GPX4 or ACSL4 on the same slice, a direct spatial correspondence and objective quantitative assessment standard between ferrous ion content and ferroptosis degree are established. At the same time, glycine quenching effectively reduces the autofluorescence background generated by aldehyde fixation, ensuring a high signal-to-noise ratio and accurate results. It is applicable to various types of samples, including paraffin sections, frozen sections, and cell samples, and has broad clinical application value. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0015] Figure 1 This is a flowchart of a method for detecting ferrous ions by fluorescent staining provided by the present invention.

[0016] Figure 2 The flowchart describes a process of removing paraffin embedding agent by heating and incubating in a dewaxing solution, followed by gradient ethanol hydration and washing with distilled water, then microwave repair with citrate buffer, and natural cooling to room temperature to obtain tissue sections with fully exposed ferrous ion binding sites.

[0017] Figure 3 This is a flowchart illustrating the process of incubating samples with glycine solution at room temperature to extinguish the autofluorescence background generated by aldehyde fixation, followed by washing with PBS to obtain pre-processed slices with reduced background signal.

[0018] Figure 4 The flowchart describes a process of incubating a staining working solution containing a fluorescent probe, ascorbic acid reducing agent, HEPES buffer, and surfactant in the dark to allow the probe to specifically bind to ferrous ions in the tissue. After washing with PBS, tissue sections with ferrous ion fluorescent signals are obtained.

[0019] Figure 5 The flowchart describes a process of obtaining double-stained sections containing both ferrous ion fluorescence signals and ferroptosis marker fluorescence signals by blocking with goat serum, sequentially adding primary antibody for overnight incubation, incubating with fluorescent secondary antibody in the dark, washing with PBS, and then obtaining double-stained sections.

[0020] Figure 6 The flowchart describes how cell nuclei are counterstained by incubating with DAPI staining solution in the dark, followed by washing with PBS and mounting with anti-fluorescence quenching mounting medium to obtain complete stained sections that can be preserved for a long time.

[0021] Figure 7 This is a flowchart illustrating the process of acquiring ferrous ions, ferroptosis markers, and nuclear fluorescence signals from cell sections using a multi-channel laser confocal microscope, processing the images with image analysis software, and obtaining quantitative values ​​of ferrous ion fluorescence intensity and colocalization coefficients.

[0022] Figure 8 It is a flowchart that compares quantitative values ​​and colocation coefficients with the normal control group to obtain the results of determining whether ferroptosis is positive, suspicious, or negative.

[0023] Figure 9 This is a schematic diagram of a ferrous ion fluorescent staining detection kit provided by the present invention.

[0024] In the figure: 1-ferrous ion fluorescent probe, 2-tissue processing solution, 3-blocking solution, 4-washing solution, 5-antibody dilution solution, 6-nuclear staining solution, 7-mounting tablet. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0026] Please see Figures 1 to 8 In a first aspect, the present invention provides a method for detecting ferrous ions by fluorescent staining, comprising the following steps: S1 was dewaxed by heating and incubating with dewaxing solution to remove paraffin embedding agent. After gradient ethanol hydration and washing with distilled water, it was repaired by microwave with citrate buffer and cooled naturally to room temperature to obtain tissue sections with fully exposed ferrous ion binding sites. S11 Place the paraffin-embedded tissue sections in dewaxing solution and incubate at 55-60℃ for 15-30 minutes to remove the paraffin embedding agent; S12 The dewaxed sections were sequentially treated with 100% ethanol, 95% ethanol, 70% ethanol and distilled water for 5 minutes each for gradient hydration. S13 involves hydrating the gradient sections with citrate buffer at pH 6.0 and then microwave-treating them at 95-100℃ for 15-20 minutes. After retrieval, the sections are allowed to cool naturally to room temperature, resulting in tissue sections with fully exposed ferrous ion binding sites.

[0027] Specifically, paraffin-embedded tissue sections are placed in dewaxing solution and incubated at 55-60℃ for 15-30 minutes. Heating melts the paraffin embedding agent, removing it from the tissue sections, as paraffin physically hinders the contact between the fluorescent probe and ferrous ions in the tissue. The dewaxed sections are then subjected to a gradient hydration process, sequentially treated with 100% ethanol, 95% ethanol, 70% ethanol, and distilled water for 5 minutes each. This gradually reduces the concentration of organic solvents in the tissue, preventing tissue shrinkage or morphological damage caused by a rapid change in osmotic pressure when directly transferring from a high-concentration organic solvent to an aqueous phase, allowing the tissue to gradually return to a hydrated state. The graded hydrated sections are then treated with pH... Citrate buffer 6.0 was microwaved at 95-100℃ for 15-20 minutes. The heat generated by the microwave and the low pH environment provided by the citric acid worked synergistically to break the methylene bridge cross-linking structure formed between the amino groups of the protein during formaldehyde fixation, restoring the protein's natural three-dimensional conformation and exposing the ferrous ion binding sites that were masked by cross-linking. After repair, the tissue was naturally cooled to room temperature to avoid the impact of sudden temperature changes on the tissue morphology, and finally, tissue sections with fully exposed ferrous ion binding sites and intact morphology were obtained.

[0028] S2 was incubated with glycine solution at room temperature to extinguish the autofluorescence background generated by aldehyde fixation in the sample. After washing with PBS, a pre-treated section with reduced background signal was obtained. S21. Tissue sections are placed in a 0.1-0.3M glycine solution and incubated at room temperature for 20-30 minutes to quench the autofluorescence background generated by aldehyde fixatives. S22 washed the quenched slices three times with PBS buffer for 5 minutes each time to obtain pre-treated slices with reduced background signal.

[0029] Specifically, the pretreated tissue sections are incubated in a 0.1-0.3M glycine solution at room temperature for 20-30 minutes. The abundant free amino groups in glycine preferentially react with the free aldehyde groups remaining in the tissue from aldehyde fixatives such as formaldehyde, forming a stable product without fluorescence. This competitively blocks or decomposes the Schiff base structure, which originally has autofluorescence properties, formed by the reaction of aldehyde groups with protein amino groups, effectively quenching the endogenous autofluorescence background and significantly improving the signal-to-noise ratio and sensitivity of subsequent fluorescence detection. The quenched sections are then washed three times with PBS buffer for 5 minutes each time to thoroughly remove residual glycine solution and reaction products, avoiding interference from residues on subsequent fluorescent probe staining, ultimately resulting in pretreated sections with significantly reduced background signal.

[0030] S3 involves incubating the tissue with a staining working solution containing a fluorescent probe, ascorbic acid reducing agent, HEPES buffer, and surfactant in the dark, allowing the probe to specifically bind to ferrous ions in the tissue. After washing with PBS, tissue sections with ferrous ion fluorescent signals are obtained. S31 The pretreated sections were placed in a staining working solution containing a ferrous ion-specific fluorescent probe, ascorbic acid reducing agent, HEPES buffer at pH 7.2-7.6 and 0.05%-0.1% Triton X-100, and incubated at 37°C in the dark for 30-60 minutes to allow the probe to specifically bind to ferrous ions in the tissue and generate a fluorescent signal. S32 washed the stained sections three times with PBS buffer for 5 minutes each time to obtain tissue sections with ferrous ion fluorescence signals.

[0031] Specifically, the pretreated sections, after endogenous fluorescence quenching, were placed in a staining working solution containing a ferrous ion-specific fluorescent probe, ascorbic acid reducing agent, HEPES buffer (pH 7.2-7.6), and 0.05%-0.1% Triton X-100 surfactant. The solution was incubated at 37°C in the dark for 30-60 minutes. The ferrous ion-specific fluorescent probe is based on a coumarin derivative structure and contains an N-pyridine oxide group. The nitrogen and oxygen atoms in this group can react with Fe... 2+ A specific coordination reaction occurs, and the probe itself is in a fluorescence quenching state while binding Fe. 2+ The intramolecular charge transfer is subsequently suppressed, resulting in a 10-50 fold increase in fluorescence intensity, thus achieving highly sensitive detection of ferrous ions; the standard reduction potential of ascorbic acid reducing agent is +0.77V, sufficient to drive Fe... 3+ / Fe 2+ The reduction reaction of the redox couple removes the Fe atoms oxidized during the formaldehyde fixation process. 3+ In-situ reduction to Fe that can be detected by probe 2+ This effectively improves the detection sensitivity of ferrous ions; the HEPES buffer maintains a stable neutral environment within the pH range of 7.2-7.6, ensuring the chemical stability of the probe molecules and the reducing activity of ascorbic acid; Triton X-100, as a nonionic surfactant, can gently embed into the cell membrane lipid bilayer at a concentration of 0.05%-0.1%, increasing membrane permeability without disrupting the overall tissue morphology, allowing the probe molecules to effectively penetrate into unstable iron pools and subcellular structures such as mitochondria in the cytoplasm, reacting with free Fe. 2+ Sufficient contact and binding to generate fluorescence signal; after incubation, the stained sections are washed three times with PBS buffer for 5 minutes each time to remove unbound free probe molecules and staining working solution residues, reduce non-specific background fluorescence, and finally obtain tissue sections with specific ferrous ion fluorescence signal.

[0032] After blocking with goat serum, S4 was incubated overnight with primary antibody, then incubated with fluorescent secondary antibody in the dark, and washed with PBS to obtain a double-stained section with both ferrous ion fluorescence signal and ferroptosis marker fluorescence signal. S41 After blocking the slides with 5%-10% normal goat serum at room temperature for 30 minutes, add GPX4 or ACSL4 primary antibody working solution and incubate overnight at 4°C. Wash with PBS 3 times, 5 minutes each time. S42 After incubation with primary antibody, the slides were added to fluorescently labeled secondary antibody working solution and incubated at 37°C in the dark for 45-60 minutes. The slides were then washed three times with PBS for 5 minutes each time to obtain double-stained slides with both ferrous ion fluorescence signals and ferroptosis marker fluorescence signals.

[0033] Specifically, tissue sections with ferrous ion fluorescence signals are blocked with 5%-10% normal goat serum at room temperature for 30 minutes. The various immunoglobulins in the serum pre-occupy Fc receptors, charged protein sites, and other non-specific protein binding sites in the tissue sections, forming a physical blocking layer to prevent the subsequent addition of primary antibodies from non-specifically binding through hydrophobic interactions or charge adsorption. After blocking, GPX4 or ACSL4 primary antibody working solution is added and incubated overnight at 4°C to allow the primary antibody to specifically bind to the target antigen in the tissue. GPX4 is the only intracellular enzyme capable of reducing lipid peroxides, and its downregulation is an important marker of ferroptosis. ACSL4 is responsible for activating polyunsaturated fatty acids to acyl-CoA to promote their incorporation into membrane phospholipids; its upregulation drives lipid peroxidation and thus promotes ferroptosis. The binding of the primary antibody to the antigen... Based on the spatial complementarity and affinity between the antibody variable region and the antigen epitope, the primary antibody was washed three times with PBS buffer for 5 minutes each time to remove unspecifically bound molecules. Then, fluorescently labeled secondary antibody working solution was added and incubated at 37°C in the dark for 45-60 minutes. Species-specific recognition between the Fc fragment of the secondary antibody and the Fc fragment of the primary antibody led to binding. One primary antibody molecule can bind to multiple secondary antibody molecules, achieving signal amplification. The fluorescent dye carried by the secondary antibody has an excitation wavelength of 590 nm and an emission wavelength of 617 nm, exhibiting red fluorescence, which forms a complementary color contrast with the green fluorescence of the ferrous ion probe, enabling spectral separation in multi-channel imaging. The secondary antibody was washed three times with PBS buffer for 5 minutes each time to remove unbound secondary antibody molecules, ultimately resulting in a double-stained section with both green ferrous ion fluorescence and red ferroptosis marker fluorescence signals on the same tissue section.

[0034] S5 was counterstained by incubation with DAPI staining solution in the dark, followed by washing with PBS and mounting with anti-fluorescence quenching mounting medium 7 to obtain a complete stained section that can be preserved for a long time. S51 Double-stained sections were incubated with 1 μg / mLDAPI staining solution at room temperature in the dark for 5-10 minutes for nuclear counterstaining, followed by washing twice with PBS for 3 minutes each time; S52 Add anti-fluorescence quenching mounting medium 7 to the counterstained section, cover with a coverslip and seal the edges, and store at 4°C in the dark to obtain a complete stained section that can be preserved for a long time.

[0035] Specifically, double-stained sections simultaneously displaying ferrous ion and ferroptosis marker fluorescence signals were incubated with 1 μg / mL DAPI staining solution at room temperature in the dark for 5-10 minutes. DAPI, a blue fluorescent dye that specifically binds to the AT-rich region in the minor groove of double-stranded DNA, exhibits electrostatic attraction between the positive charge in its molecule and the negative charge of the DNA phosphate backbone. After binding, it intercalates between base pairs, significantly increasing its fluorescence quantum yield and producing a bright blue fluorescent signal. This provides a clear morphological reference for the cell nuclei in the tissue sections, facilitating the determination of the spatial distribution of ferrous ions and ferroptosis markers at the cellular and subcellular levels in subsequent analyses. The sections were washed twice with PBS buffer for 3 minutes each time to remove unbound free DAPI molecules. To avoid interference from background fluorescence, anti-fluorescence quenching mounting medium 7 was added to the counterstained sections. This mounting medium 7 contains anti-photobleaching components such as p-phenylenediamine derivatives or vitamin E analogs, which can effectively remove free radicals generated by excitation light irradiation and delay the photobleaching effect of fluorescent dyes. At the same time, the refractive index of mounting medium 7 is about 1.52, which matches the refractive index of biological tissue sections, reducing light scattering at the tissue-media interface and improving the clarity and resolution of imaging. Cover with a coverslip and seal the edges to prevent the sections from drying and oxidizing. Store at 4°C in the dark to further slow down fluorescence decay, and finally obtain complete stained sections that can be stored for a long time and have stable fluorescence signals.

[0036] The S6 uses a laser confocal microscope to acquire ferrous ions, ferroptosis markers, and nuclear fluorescence signals from slices via multiple channels. After image analysis software processing, quantitative values ​​of ferrous ion fluorescence intensity and colocalization coefficients are obtained. S61 placed the complete stained section under a laser confocal microscope or a fluorescence microscope and used a multi-channel imaging mode to acquire ferrous ion fluorescence signals, ferroptosis marker fluorescence signals and DAPI cell nuclear fluorescence signals, respectively. S62 uses image analysis software to perform semi-quantitative analysis of ferrous ion fluorescence intensity and calculates the colocation coefficient between ferrous ion fluorescence signal and ferroptosis marker fluorescence signal to obtain quantitative values ​​of ferrous ion fluorescence intensity and colocation coefficients.

[0037] Specifically, intact stained sections suitable for long-term preservation are placed under a laser confocal microscope or fluorescence microscope. A multi-channel imaging mode is used, with excitation wavelengths of 450 nm and emission wavelengths of the ferriion-specific fluorescent probe (520 nm), excitation wavelengths of the ferroptosis marker fluorescent secondary antibody (590 nm and 617 nm), and excitation wavelengths of the DAPI nuclear dye (358 nm and 461 nm). The pinhole structure of the laser confocal microscope filters out scattered light outside the focal plane, obtaining high-resolution optical images of the sections and avoiding interference between signals from different focal planes. Fluorescence signal images from the three channels are acquired sequentially or simultaneously, and spectral separation of the multicolor fluorescence is achieved using a combination of a beam splitter and bandpass filters. To avoid spectral crosstalk between channels, the acquired images were processed using image analysis software. A fluorescence intensity threshold was set to distinguish between signal and background regions. Semi-quantitative analysis of the fluorescence intensity of the ferrous ion fluorescence channel was performed, and the average or integrated fluorescence intensity was calculated as a quantitative indicator. Simultaneously, the Manders' overlap coefficient algorithm was used to calculate the co-location coefficient between the ferrous ion fluorescence signal and the ferroptosis marker fluorescence signal. This algorithm quantitatively assesses the degree of overlap between ferrous ions and ferroptosis markers in spatial distribution by calculating the proportion of co-located pixel intensity to the total pixel intensity in the two fluorescence channels, thereby determining their coexistence relationship in the same cell or subcellular structure. Finally, the quantitative value of ferrous ion fluorescence intensity and the co-location coefficient were obtained.

[0038] S7 compares the quantitative values ​​and colocation coefficients with the normal control group to obtain the results of determining whether ferroptosis is positive, suspicious, or negative.

[0039] S71 compares the quantitative values ​​of ferrous ion fluorescence intensity and colocation coefficients with the mean values ​​of the normal control group; S72 When the fluorescence intensity is more than twice the mean of the normal control group and the colocalization coefficient is greater than 0.6, it is judged as positive for ferroptosis; when the fluorescence intensity is 1.5-2 times the mean of the normal control group and the colocalization coefficient is 0.3-0.6, it is judged as suspicious for ferroptosis; when the fluorescence intensity is less than 1.5 times the mean of the normal control group and the colocalization coefficient is less than 0.3, it is judged as negative for ferroptosis, thus obtaining the judgment result of positive, suspicious or negative for ferroptosis.

[0040] Specifically, the quantitative values ​​of ferrous ion fluorescence intensity and colocation coefficients obtained in step six are compared with the mean values ​​obtained by detecting and calculating at least three normal control tissue sections under the same experimental conditions. A baseline physiological level of ferrous ions in the tissue is established using the normal control group, and statistical methods are used to reduce misjudgments caused by individual and batch-specific differences. When the ferrous ion fluorescence intensity of the test sample is more than twice the mean of the normal control group, it indicates a significant abnormal accumulation of ferrous ions in the tissue. Furthermore, if the colocation coefficient of ferrous ions with GPX4 or ACSL4 is greater than 0.6, it indicates a high spatial overlap between ferrous ion accumulation and changes in the expression of ferroptosis markers, both jointly indicating ferroptosis. If ferrous ions are detected, the sample is considered positive for ferroptosis. If the fluorescence intensity is 1.5-2 times higher than the mean of the normal control group, it indicates a moderate increase in ferrous ions, and the colocation coefficient is 0.3-0.6, indicating a limited spatial correlation between ferrous ions and ferroptosis markers. In this case, ferroptosis is considered suspicious, suggesting further confirmation by combining other detection indicators or repeating experiments. If the fluorescence intensity is 1.5 times lower than the mean of the normal control group, it indicates that ferrous ions are at normal physiological levels, and the colocation coefficient is less than 0.3, indicating no significant spatial colocation relationship between ferrous ions and ferroptosis markers. In this case, ferroptosis is considered negative. The final result is determined as positive, suspicious, or negative for ferroptosis.

[0041] Please see Figure 9 Secondly, a ferrous ion fluorescent staining detection kit for use in the ferrous ion fluorescent staining detection method described in the first aspect includes a ferrous ion fluorescent probe 1, a tissue processing solution 2, a blocking solution 3, a washing solution 4, an antibody diluent 5, a nuclear staining solution 6, and a mounting medium 7.

[0042] In this embodiment, the ferrous ion fluorescent probe 1 is a chemically modified oxynitride structure fluorescent probe with an emission wavelength of 570-590 nm, used to specifically identify and label ferrous ions in tissue sections; the tissue processing solution 2 includes dewaxing solution, gradient ethanol rehydration solution, antigen retrieval solution, and permeabilization solution; the blocking solution 3 includes a mixed solution of 5%-10% normal serum and 0.1%-0.5% Triton X-100; the kit also includes a ferroptosis marker primary antibody combination, which includes at least one of GPX4 antibody, SLC7A11 antibody, ACSL4 antibody, TFR1 antibody, and FTH1 antibody; the kit is suitable for fluorescent staining detection of ferrous ions in three sample types: paraffin-embedded tissue sections, frozen tissue sections, and cell smears.

[0043] The above-disclosed embodiments are merely one or more preferred embodiments of this application and should not be construed as limiting the scope of this application. Those skilled in the art can understand that all or part of the processes for implementing the above embodiments and equivalent changes made in accordance with the claims of this application still fall within the scope of this application.

Claims

1. A method for detecting ferrous ions by fluorescent staining, characterized in that, Includes the following steps: The paraffin embedding agent was removed by heating and incubating with dewaxing solution. After gradient ethanol hydration and washing with distilled water, the tissue sections were microwaved with citrate buffer and naturally cooled to room temperature to obtain tissue sections with fully exposed ferrous ion binding sites. The autofluorescence background generated by aldehyde fixation in the sample was extinguished by incubation with glycine solution at room temperature. After washing with PBS, pre-treated sections with reduced background signal were obtained. By incubating the tissue with a staining working solution containing a fluorescent probe, ascorbic acid reducing agent, HEPES buffer and surfactant in the dark, the probe specifically binds to ferrous ions in the tissue. After washing with PBS, tissue sections with ferrous ion fluorescent signals are obtained. After blocking with goat serum, the primary antibody was added and incubated overnight, followed by incubation with fluorescent secondary antibody in the dark. After washing with PBS, a double-stained section with both ferrous ion fluorescence signal and ferroptosis marker fluorescence signal was obtained. Cell nuclei were counterstained by incubation with DAPI staining solution in the dark, washed with PBS, and mounted with anti-fluorescence quenching mounting medium to obtain complete stained sections that can be preserved for a long time. Ferrous ion, ferroptosis markers, and nuclear fluorescence signals were acquired from sections using a multi-channel laser confocal microscope. The images were then processed by image analysis software to obtain quantitative values ​​of ferrous ion fluorescence intensity and colocalization coefficients. The quantitative values ​​and colocation coefficients were compared with the normal control group to obtain the results of determining whether ferroptosis was positive, suspicious, or negative.

2. The method for detecting ferrous ions by fluorescent staining as described in claim 1, characterized in that, The procedure described in "removing paraffin embedding agent by heating incubation with dewaxing solution, followed by gradient ethanol hydration and washing with distilled water, microwave repair with citrate buffer, and natural cooling to room temperature to obtain tissue sections with fully exposed ferrous ion binding sites" includes the following steps: Place the paraffin-embedded tissue sections in dewaxing solution and incubate at 55-60℃ for 15-30 minutes to remove the paraffin embedding agent; The dewaxed sections were sequentially treated with 100% ethanol, 95% ethanol, 70% ethanol and distilled water for 5 minutes each for gradient hydration. After gradient hydration, the sections were microwaved at 95-100℃ for 15-20 minutes with citrate buffer at pH 6.

0. After remedy, the sections were allowed to cool naturally to room temperature to obtain tissue sections with fully exposed ferrous ion binding sites.

3. The method for detecting ferrous ions by fluorescent staining as described in claim 1, characterized in that, The procedure for "inhibiting the autofluorescence background generated by aldehyde fixation in the sample by incubation with glycine solution at room temperature, followed by washing with PBS to obtain pre-treated sections with reduced background signal" includes the following steps: Tissue sections were incubated in 0.1-0.3M glycine solution at room temperature for 20-30 minutes to quench the autofluorescence background generated by aldehyde fixatives. The quenched sections were washed three times with PBS buffer for 5 minutes each time to obtain pretreated sections with reduced background signal.

4. The method for detecting ferrous ions by fluorescent staining as described in claim 1, characterized in that, The procedure described in "incubating the tissue with a staining working solution containing a fluorescent probe, ascorbic acid reducing agent, HEPES buffer, and surfactant in the dark to allow the probe to specifically bind to ferrous ions in the tissue, followed by washing with PBS to obtain tissue sections with ferrous ion fluorescent signals" includes the following steps: The pretreated sections were placed in a staining working solution containing a ferrous ion-specific fluorescent probe, ascorbic acid reducing agent, HEPES buffer (pH 7.2-7.6), and 0.05%-0.1% Triton X-100, and incubated at 37°C in the dark for 30-60 minutes to allow the probe to specifically bind to ferrous ions in the tissue and generate a fluorescent signal. The stained sections were washed three times with PBS buffer for 5 minutes each time to obtain tissue sections with ferrous ion fluorescence signals.

5. The method for detecting ferrous ions by fluorescent staining as described in claim 1, characterized in that, The procedure described in "blocking with goat serum followed by overnight incubation with primary antibody, incubation with fluorescent secondary antibody in the dark, washing with PBS, and obtaining double-stained sections with both ferrous ion fluorescence and ferroptosis marker fluorescence signals" includes the following steps: After blocking the sections with 5%-10% normal goat serum at room temperature for 30 minutes, add GPX4 or ACSL4 primary antibody working solution and incubate overnight at 4°C. Wash with PBS 3 times, 5 minutes each time. After incubation with primary antibody, the slides were added to fluorescently labeled secondary antibody working solution and incubated at 37°C in the dark for 45-60 minutes. The slides were then washed three times with PBS for 5 minutes each time to obtain double-stained slides with both ferrous ion fluorescence signals and ferroptosis marker fluorescence signals.

6. The method for detecting ferrous ions by fluorescent staining as described in claim 1, characterized in that, The procedure for "counter-staining cell nuclei by incubating with DAPI staining solution in the dark, washing with PBS, and mounting with anti-fluorescence quenching mounting medium to obtain complete stained sections that can be preserved for a long time" includes the following steps: Double-stained sections were incubated with 1 μg / mLDAPI staining solution at room temperature in the dark for 5-10 minutes for nuclear counterstaining, followed by washing twice with PBS for 3 minutes each time. After counterstaining, add anti-fluorescence quenching mounting medium to the slide, cover with a coverslip and seal the edges, and store at 4°C in the dark to obtain a complete stained slide that can be preserved for a long time.

7. A ferrous ion fluorescent staining detection kit, used in the ferrous ion fluorescent staining detection method according to any one of claims 1-6, characterized in that, It includes ferrous ion fluorescent probes, tissue processing solutions, blocking solutions, washing solutions, antibody diluents, nuclear staining solutions, and mounting media.

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