Methods for detecting neuronal death coupled with fluorescence and electron microscopy

Abstract

The application belongs to the field of neurology and provides a method for detecting neuron death by coupling fluorescence and electron microscopy, comprising the following steps: step one, using Pulsineli's four-vessel occlusion method to make a rat transient global ischemia model, and sampling and processing the model animal to ensure the stability of the sample during the experiment; step two, performing monochromatic fluorescence staining on the sample obtained in step one, then converting the Rhodamine R6 fluorescent label in the slice into 3,3'-diaminobenzidine precipitation, and making the slice into an electron microscopy sample, and determining the experimental results. The application first applies Rhodamine R6 to the field of ultra-morphological detection of neuron death, converts the fluorescent signal of Rhodamine R6 into diaminobenzidine precipitation, thereby realizing the coupling of fluorescence and electron microscopy, and observing the ultra-localization of the positive substance in the dead cells by using an electron microscope.

Description

Technical Field

[0001] This invention belongs to the field of neuroscience, and in particular relates to a method for detecting neuronal death by coupled fluorescence and electron microscopy. Background Technology

[0002] Neuronal death is one of the main pathological features of many acute and chronic neurodegenerative diseases, such as cerebral ischemia, hypoxia, and epilepsy. Neuronal death visualization technology can not only qualitatively and / or quantitatively analyze the occurrence and development of neuronal death, but also be used to evaluate the success of animal models or the effectiveness of neuroprotective drugs or measures.

[0003] To date, morphological characteristics remain one of the main criteria for distinguishing between dying or dying cells. Therefore, optical microscopy and electron microscopy play crucial roles in the study of neuronal death. Optical microscopy is more convenient and practical than electron microscopy because it is less time-consuming and less expensive. However, only electron microscopy can observe the ultra-fine details of neuronal death, making its importance irreplaceable to date. Commonly used histochemical techniques for detecting neuronal death include hematoxylin-eosin (HE) staining, Nissl staining, acid fuchsin staining, Fluoro-Jade (FJ) staining, and silver staining. However, these methods are often difficult to use for double or multiple staining, making it difficult to determine the type of dead cells without referring to the morphological and locational characteristics of positive cells. Furthermore, the positive structures labeled by these techniques can only be observed under an optical microscope and cannot be further verified and studied using electron microscopy.

[0004] Rhodamine R6 is a lipid-soluble rhodamine derivative widely used in fluorescence microscopy or flow cytometry to monitor mitochondrial membrane potential in living cells (e.g., Invitrogen™ R648MP). This dye is rarely used in in vivo animal experiments, and to date, there are no reports on its potential for ultramorphological detection of neuronal death. Summary of the Invention

[0005] The purpose of this invention is to provide a method for coupled detection of neuronal death using fluorescence and electron microscopy, aiming to solve the problem that conventional histochemical detection techniques for neuronal death are difficult to simultaneously utilize for fluorescence and electron microscopy observation. Rhodamine R6 is a lipid-soluble rhodamine derivative widely used in fluorescence microscopy or flow cytometry to monitor mitochondrial membrane potential in living cells (e.g., Invitrogen™ R648MP). This dye is rarely used in in vivo animal experiments, and to date, there are no reports on its suitability for ultramorphological detection of neuronal death.

[0006] The present invention is implemented as follows: First, the slide specimen is stained with monochromatic fluorescence. Then, the Rhodamine R6 fluorescent label in the slide is converted into 3,3'-diaminobenzidine precipitate. The slide is then prepared as an electron microscopy specimen, and the experimental results are measured.

[0007] The method for detecting neuronal death using fluorescence and electron microscopy coupling includes the following steps:

[0008] Step 1: A rat model of transient global cerebral ischemia was established using Pulsinelli's four-vessel occlusion method. Samples were taken from the model animals to ensure the stability of the specimens during the experiment.

[0009] Step 2: The specimens obtained in Step 1 were stained with monochromatic fluorescence. Then, the Rhodamine R6 fluorescent label in the sections was converted into 3,3'-diaminobenzidine precipitate. The sections were then prepared as electron microscopy specimens, and the experimental results were measured.

[0010] As a further embodiment of the present invention, the rat global cerebral ischemia model and sampling process described in step one using Pulsinelli's four-vessel occlusion method include the following steps:

[0011] S1. Anesthetize rats by intraperitoneal injection of 10% chloral hydrate at a ratio of 0.4 ml / 100 g. Fix the rats in a supine position, wipe the skin in the middle of the rat's neck with an alcohol swab and trim the rat hair, and disinfect with povidone-iodine.

[0012] S2. Make a 2cm incision in the middle of the rat's neck, and separate the sternocleidomastoid muscle outward along the medial border of the sternocleidomastoid muscle. Separate the fascia around the common carotid artery and free a section of blood vessel for threading. Then treat the contralateral artery in the same way. After completion, suture the skin and disinfect with povidone-iodine.

[0013] S3. Fix the rat in a prone position, disinfect it using the method described in S1, make a 1cm incision in the middle of the back of the neck, use blunt forceps to separate the paravertebral muscles from the middle to expose the first cervical vertebral lamina, and then move it outward and upward to expose the pterygoid foramina on both sides.

[0014] S4. Insert the electrocoagulator into the wing hole to perform electrocoagulation. After electrocoagulation, suture the muscle and skin, wipe with iodine for disinfection, and put it back in the cage.

[0015] S5. Wait 24 hours, remove the rat, fix it in a supine position, expose the common carotid arteries on both sides using the method described in S2, clamp them with arterial clamps, and then release the fixation. The disappearance of the righting reflex and corneal reflex in the rat within 15-30 seconds proves that the whole brain ischemia was successful. Discard rats that have convulsions during the process.

[0016] S6. After clamping both common carotid arteries for 13 minutes, remove the arterial clamps to restore blood flow; then suture the skin, disinfect with povidone-iodine, and return the animal to its cage for continued feeding.

[0017] S7. 1 to 14 days after ischemia-reperfusion, the experimental animals were anesthetized with 10% chloral hydrate, the thoracic cavity was opened to expose the heart, and the perfusion pump was used to operate through the ascending aorta. The blood was first flushed with physiological saline, and then 4% paraformaldehyde was used for perfusion fixation.

[0018] S8. After perfusion, remove the animal's brain and place it in 4% paraformaldehyde for 4-6 hours. Then, preserve the brain tissue in 5% sucrose.

[0019] S9. Use a vibratory slicer to cut brain tissue into sections 30-50 μm thick, wash three times with 0.1 M phosphate buffer for 5-15 minutes each time, and store at 4°C.

[0020] As a further embodiment of the present invention, the method for detecting neuronal death by coupled fluorescence and electron microscopy in step two includes the following steps:

[0021] S1. Collect the slices into a 24-well plate and immerse them in a 0.5-3% hydrogen peroxide solution. React on a shaker for 10-30 minutes.

[0022] S2. Add a potassium permanganate aqueous solution with a concentration of 0.06~0.25% and a pH of 4.5~7.5 to pretreat the sections for 5~15 minutes. Potassium permanganate pretreatment can significantly reduce the staining background. Omitting this step has no significant impact on the experimental results.

[0023] S3. Briefly rinse the pretreated slides with distilled water, then rinse three times with 0.1M PB buffer for 5-10 minutes each time.

[0024] S4. Add Rhodamine R6 working solution and incubate at room temperature in the dark for 5 min to 2 h. The negative control group is replaced with 0.1 M phosphate buffer instead of Rhodamine R6.

[0025] S5. Wash the incubated slices three times with 0.1M PB buffer, each time for 5-10 minutes;

[0026] S6. Some sections were stained using the FluoroJade B (FJ-B) staining procedure to confirm neuronal death, while others were stained with 4',6-diamidinyl-2-phenylindole to confirm changes in nuclear morphology. After staining, the sections were transferred to glass slides and observed and photographed using a fluorescence microscope.

[0027] S7. After observation under a fluorescence microscope, the Rhodamine R6 stained sections were placed back into the 24-well plate and incubated at room temperature for 0.5 to 1.0 hours in 0.1M phosphate buffer containing 4-10% normal goat serum and 0.05-0.1% polyethylene glycol octylphenyl ether.

[0028] S8. Discard the solution from S7, add mouse monoclonal anti-rhodamine antibody, and incubate overnight at 4°C.

[0029] S9. Wash the sections three times with 0.1M phosphate buffer, add goat anti-mouse IgG H&L (Biotin) secondary antibody, and incubate at room temperature for 1-2 hours;

[0030] S10. Wash the sections three times with 0.1M phosphate buffer for 5-15 minutes each time, then add the avidin-biotin-peroxidase complex and react at room temperature for 0.5-1.5 hours.

[0031] S11. Wash the reacted sections three times with 0.1M phosphate buffer for 5-15 minutes each time, and then wash with 50mM Tris buffer at pH 7.4 for 5-15 minutes.

[0032] S12. Add 50 mM Tris buffer containing 0.001~0.05% 3,3'-diaminobenzidine and react at room temperature for about 5~15 minutes.

[0033] S13. Add 50mM Tris buffer containing 0.005~0.03% hydrogen peroxide and 0.001~0.05% 3,3'-diaminobenzidine, and react at room temperature for about 5~15 minutes.

[0034] S14. First, wash the slides with 50mM Tris buffer at pH 7.4 for 5-15 minutes, then wash the slides three times with 0.1M phosphate buffer for 5-15 minutes each time. Finally, add 0.1M phosphate buffer at pH 4.5-7.4 containing 0.75-1.00% osmium tetroxide and react at 4°C for 0.5-2 hours.

[0035] S15. Perform gradient dehydration on the sections according to concentrations of 50%, 70%, 80%, 90%, 95%, 100% I, and 100% II; add propylene oxide to replace ethanol for 5-15 minutes, and repeat 1-2 times.

[0036] S16. Weigh Epon812 embedding agent, dodecenylsuccinic anhydride, methylnonylacetaldehyde, and 2,4,6-tris(dimethylaminomethyl)phenol, prepare the embedding solution according to the ordinary electron microscope preparation method, and add the section to a 1:1 volume ratio mixture of propylene oxide and embedding solution, and incubate overnight at room temperature.

[0037] S17. Place the slices into a plastic embedding container, add embedding solution, react at 40°C for 1 day, and then react at 60°C for 2 days to allow the embedding solution to fully polymerize.

[0038] S18. Using an ultramicrotome, the embedded specimens were prepared into ultrathin sections with a thickness of 80-120 nm and collected on a copper grid. The samples from the ischemia / reperfusion group stained with 3,3'-diaminobenzidine were observed directly under an electron microscope after ultrathin sectioning.

[0039] Ultrathin sections of S19, the sham-operated group, and the negative control group (with Rhodamine R6 omitted) were stained with 2.0–6.0% uranyl acetate for 3 minutes, then washed three times with distilled water for 2 minutes each time. Next, the ultrathin sections were stained with 0.1–0.5% lead citrate for 2.5 minutes, then washed three times with distilled water for 2 minutes each time. After drying, they were used for electron microscopy.

[0040] The method for detecting neuronal death using fluorescence and electron microscopy coupled in this invention has the following beneficial effects:

[0041] This invention is the first to apply Rhodamine R6 to the field of ultramorphological detection of neuronal death. The fluorescence signal of Rhodamine R6 is converted into diaminobenzidine precipitation, thereby achieving the coupling of fluorescence and electron microscopy, and using electron microscopy to observe the ultra-micro localization of positive substances in dead cells. Attached Figure Description

[0042] Figure 1 Image showing the results of double fluorescence staining of rat hippocampus with FluoroJade-B (FJ-B) and Rhodamine R6;

[0043] Figure 2 The image shows the results of FJ-B and Rhodamine R6 staining in the CA1 region of the rat hippocampus, as a confocal dual fluorescence image.

[0044] Figure 3 A high-resolution confocal image showing the granular distribution of Rhodamine R6-positive structures in the CA1 region of the rat hippocampus;

[0045] Figure 4 A confocal image showing a map of degenerated neurons specifically labeled with Rhodamine R6;

[0046] Figure 5 Fluorescence and optical microscopy images of the Rhodamine R6 fluorescence signal converted into peroxidase / DAB reaction products;

[0047] Figure 6 This is an electron microscope image showing the distribution of Rhodamine R6 fluorescence converted to DAB / peroxidase reaction products in CA1 neurons of the sham-operated group;

[0048] Figure 7 This is an electron micrograph showing the distribution of Rhodamine R6 fluorescence converted to peroxidase / DAB reaction products in CA1 neurons of the 2-day ischemia / reperfusion group;

[0049] Figure 8 The image shows two severely damaged CA1 neurons after ischemia-reperfusion, as displayed by an electron microscope. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0051] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0052] A method for detecting neuronal death using fluorescence and electron microscopy coupled together, the method comprising the following steps:

[0053] Step 1: A rat model of transient global cerebral ischemia was established using Pulsinelli's four-vessel occlusion method. Samples were taken from the model animals to ensure the stability of the specimens during the experiment.

[0054] Step 2: The specimens obtained in Step 1 were stained with monochromatic fluorescence. Then, the Rhodamine R6 fluorescent label in the sections was converted into 3,3'-diaminobenzidine precipitate. The sections were then prepared as electron microscopy specimens, and the experimental results were measured.

[0055] As a further embodiment of the present invention, the rat global cerebral ischemia model and sampling process described in step one using Pulsinelli's four-vessel occlusion method include the following steps:

[0056] S1. Anesthetize rats by intraperitoneal injection of 10% chloral hydrate at a ratio of 0.4 ml / 100 g. Fix the rats in a supine position, wipe the skin in the middle of the rat's neck with an alcohol swab and trim the rat hair, and disinfect with povidone-iodine.

[0057] S2. Make a 2cm incision in the middle of the rat's neck, and separate the sternocleidomastoid muscle outward along the medial border of the sternocleidomastoid muscle. Separate the fascia around the common carotid artery and free a section of blood vessel for threading. Then treat the contralateral artery in the same way. After completion, suture the skin and disinfect with povidone-iodine.

[0058] S3. Fix the rat in a prone position, disinfect it using the method described in S1, make a 1cm incision in the middle of the back of the neck, use blunt forceps to separate the paravertebral muscles from the middle to expose the first cervical vertebral lamina, and then move it outward and upward to expose the pterygoid foramina on both sides.

[0059] S4. Insert the electrocoagulator into the wing hole to perform electrocoagulation. After electrocoagulation, suture the muscle and skin, wipe with iodine for disinfection, and put it back in the cage.

[0060] S5. Wait 24 hours, remove the rat, fix it in a supine position, expose the common carotid arteries on both sides using the method described in S2, clamp them with arterial clamps, and then release the fixation. The disappearance of the righting reflex and corneal reflex in the rat within 15-30 seconds proves that the whole brain ischemia was successful. Discard rats that have convulsions during the process.

[0061] S6. After clamping both common carotid arteries for 13 minutes, remove the arterial clamps to restore blood flow; then suture the skin, disinfect with povidone-iodine, and return the animal to its cage for continued feeding.

[0062] S7. 1 to 14 days after ischemia-reperfusion, the experimental animals were anesthetized with 10% chloral hydrate, the thoracic cavity was opened to expose the heart, and the perfusion pump was used to operate through the ascending aorta. The blood was first flushed with physiological saline, and then 4% paraformaldehyde was used for perfusion fixation.

[0063] S8. After perfusion, remove the animal's brain and place it in 4% paraformaldehyde for 4-6 hours. Then, preserve the brain tissue in 5% sucrose.

[0064] S9. Use a vibratory slicer to cut brain tissue into sections 30-50 μm thick, wash three times with 0.1 M phosphate buffer for 5-15 minutes each time, and store at 4°C.

[0065] As a further embodiment of the present invention, the method for detecting neuronal death by coupled fluorescence and electron microscopy in step two includes the following steps:

[0066] S1. Collect the slices into a 24-well plate and immerse them in a 0.5-3% hydrogen peroxide solution. React on a shaker for 10-30 minutes.

[0067] S2. Add a potassium permanganate aqueous solution with a concentration of 0.06~0.25% and a pH of 4.5~7.5 to pretreat the sections for 5~15 minutes. Potassium permanganate pretreatment can significantly reduce the staining background. Omitting this step has no significant impact on the experimental results.

[0068] S3. Briefly rinse the pretreated slides with distilled water, then rinse three times with 0.1M PB buffer for 5-10 minutes each time.

[0069] S4. Add Rhodamine R6 working solution and incubate at room temperature in the dark for 5 min to 2 h. The negative control group is replaced with 0.1 M phosphate buffer instead of Rhodamine R6.

[0070] S5. Wash the incubated slices three times with 0.1M PB buffer, each time for 5-10 minutes;

[0071] S6. Some sections were stained according to the FluoroJade B (FJ-B) staining procedure to confirm neuronal death, and some sections were stained with 4',6-diamidinyl-2-phenylindole to confirm changes in cell nuclear morphology. After staining, the sections were picked up and placed on a glass slide for observation and photography using a fluorescence microscope.

[0072] S7. After observation under a fluorescence microscope, the Rhodamine R6 stained sections were placed back into the 24-well plate and incubated at room temperature for 0.5 to 1.0 hours in 0.1M phosphate buffer containing 4-10% normal goat serum and 0.05-0.1% polyethylene glycol octylphenyl ether.

[0073] S8. Discard the solution from S7, add mouse monoclonal anti-rhodamine antibody, and incubate overnight at 4°C.

[0074] S9. Wash the sections three times with 0.1M phosphate buffer, add goat anti-mouse IgG H&L (Biotin) secondary antibody, and incubate at room temperature for 1-2 hours;

[0075] S10. Wash the sections three times with 0.1M phosphate buffer for 5-15 minutes each time, then add the avidin-biotin-peroxidase complex and react at room temperature for 0.5-1.5 hours.

[0076] S11. Wash the reacted sections three times with 0.1M phosphate buffer for 5-15 minutes each time, and then wash with 50mM Tris buffer at pH 7.4 for 5-15 minutes.

[0077] S12. Add 50 mM Tris buffer containing 0.001~0.05% 3,3'-diaminobenzidine and react at room temperature for about 5~15 minutes.

[0078] S13. Discard the solution in S12, add 50mM Tris buffer containing 0.005~0.03% hydrogen peroxide and 0.001~0.05% 3,3'-diaminobenzidine, and react at room temperature for about 5~15 minutes.

[0079] S14. First, wash the slides with 50mM Tris buffer at pH 7.4 for 5-15 minutes, then wash the slides three times with 0.1M phosphate buffer for 5-15 minutes each time. Finally, add 0.1M phosphate buffer at pH 4.5-7.4 containing 0.75-1.00% osmium tetroxide and react at 4°C for 0.5-2 hours.

[0080] S15. Perform gradient dehydration on the sections according to concentrations of 50%, 70%, 80%, 90%, 95%, 100% I, and 100% II, and add propylene oxide to replace ethanol for 5-15 minutes, repeating 1-2 times.

[0081] S16. Weigh Epon812 embedding agent, dodecenylsuccinic anhydride, methylnonylacetaldehyde, and 2,4,6-tris(dimethylaminomethyl)phenol, prepare the embedding solution according to the ordinary electron microscope preparation method, and add the section to a 1:1 volume ratio mixture of propylene oxide and embedding solution, and incubate overnight at room temperature.

[0082] S17. Place the slices into a plastic embedding container, add embedding solution, react at 40°C for 1 day, and then react at 60°C for 2 days to allow the embedding solution to fully polymerize.

[0083] S18. Using an ultramicrotome, the embedded specimens were prepared into ultrathin sections with a thickness of 80-120 nm and collected on a copper grid. The samples from the ischemia / reperfusion group stained with 3,3'-diaminobenzidine were observed directly under an electron microscope after ultrathin sectioning.

[0084] Ultrathin sections of S19, the sham-operated group, and the negative control group (with Rhodamine R6 omitted) were stained with 2.0–6.0% uranyl acetate for 3 minutes, then washed three times with distilled water for 2 minutes each time. Next, the ultrathin sections were stained with 0.1–0.5% lead citrate for 2.5 minutes, then washed three times with distilled water for 2 minutes each time. After drying, they were used for electron microscopy.

[0085] As a further embodiment of the present invention, the Rhodamine R6 is prepared into a stock solution with a concentration of 1.0 mg / ml using ethanol or dimethyl sulfoxide (DMSO) and stored at -20°C. Before use, it is prepared into a working solution with buffer solution at a concentration of 500~5000×.

[0086] Experimental Results Observation:

[0087] Rhodamine R6 has a maximum absorption excitation wavelength of 528 nm and a maximum emission wavelength of 551 nm. It can be detected using TRITC excitation and emission filters; red cells indicate positive degenerated neurons. DAB-stained sections were observed using a conventional optical microscope; electron microscopy specimens were observed using a transmission electron microscope.

[0088] This invention uses a transient global cerebral ischemia-reperfusion animal model, combined with Figure 1 It can be seen that after the successful creation of the model was verified by FluoroJade B (FJ-B) staining, neurons in the CA1 region of the hippocampus died.

[0089] exist Figure 1 In the diagram, A1-C1 is FJ-B staining; A2-C2 is Rhodamine R6 staining; A3-C3 are composite diagrams of A1-C1 and A2-C2, respectively.

[0090] In the sham-operated group of mice, no positive cells were observed in the hippocampus for FJ-B (A1) and Rhodamine R6 (A2) staining (A1-A3). FJ-B staining results at 3 and 14 days of ischemia / reperfusion (B1–C1) indicated persistent progressive neuronal death in the CA1 region of the hippocampus. Similar to FJ-B staining results, the affinity of CA1 neurons for Rhodamine R6 (B2–C2) was also significantly enhanced. Dual fluorescence staining showed that Rhodamine R6-positive cells were almost identical to FJ-B-positive cells in terms of both timing and spatial distribution.

[0091] Combination Figure 2 It can be seen that the dual fluorescent labeling of FJ-B and Rhodamine indicates that the Rhodamine R6 staining results are almost completely consistent with those of FJ-B;

[0092] exist Figure 2In the figures, A1 and A2 show the staining results of FJ-B and Rhodamine R6 in the CA1 region of the hippocampus in the sham-operated group, respectively; A3 is a composite image of A1 and A2; B1-D1 show the staining results of FJ-B in the CA1 region of the hippocampus at 2, 7, and 14 days after ischemia / reperfusion, respectively; B2-D2 show the staining results of Rhodamine R6 in the CA1 region of the hippocampus at 2, 7, and 14 days after ischemia / reperfusion, respectively; and B3-D3 are composite images of B1-D1 and B2-D2, respectively.

[0093] Compared with the sham-operated group, the affinity of CA1 neurons in ischemic / reperfusion rats for FJ-B and Rhodamine R6 was significantly enhanced. All FJ-B positive cells showed Rhodamine R6 positive staining, and vice versa.

[0094] Combination Figure 3 It can be seen that the high-resolution confocal images further show that the Rhodamine R6 positive structures in the CA1 region of the rat hippocampus are distributed in a granular manner;

[0095] exist Figure 3 In the images, A1 and A2 show the Rhodamine R6 and DAPI staining results in the CA1 region of the hippocampus of the sham-operated group; A3 is a composite image of A1 and A2; B1-B3 are magnified images of the area within the square frame in A1-A3. C1 and C2 show the Rhodamine R6 and DAPI staining results in the CA1 region of the hippocampus of rats 2 days after ischemia / reperfusion; B3 is a composite image of B1 and B2; D1-D3 are magnified images of the area within the square frame in C1-C3.

[0096] Compared with the sham surgery group (A1 and B1), the ischemia-reperfusion group showed a significant increase in granular material in the cytoplasm of CA1 neurons in the hippocampus (C1 and D1).

[0097] Combination Figure 4 It can be seen that if 0.1M phosphate buffer is used instead of Rhodamine R6 in the staining process, no positive staining will be observed.

[0098] exist Figure 4 In the images, A and B are photographs of the CA1 region of the hippocampus in the sham-operated group. In the staining procedure of image A, 0.1M phosphate buffer was used instead of Rhodamine R6, and no fluorescent staining was observed in the CA1 region. Image B shows Rhodamine R6 staining, in which the CA1 region of the sham-operated group shows diffuse staining, with no obviously positive neurons.

[0099] C and D are photographs of the CA1 region in rats in the 2-day ischemia / reperfusion group. In the staining procedure of image C, 0.1M phosphate buffer was used instead of Rhodamine R6, and no fluorescent staining was observed in the CA1 region; image D shows Rhodamine R6 staining, in which many positive neurons appeared in the CA1 region.

[0100] This invention converts the Rhodamine R6 fluorescence signal into a DAB / peroxidase reaction product, and binds... Figure 5 It can be seen that optical and fluorescence microscopy observations show that, in both the sham surgery group and the ischemia / reperfusion group, the distribution of peroxidase / DAB reaction products and the fluorescence signal of Rhodamine R6 is almost completely consistent.

[0101] exist Figure 5 In the images, A1 is a fluorescence image of the CA1 region of the hippocampus stained with Rhodamine R6 in the sham-operated group; A2 is a light micrograph of the product of the DAB / peroxidase reaction after converting the Rhodamine R6 fluorescence signal in the sham-operated group; and A3 shows that 0.1M phosphate buffer was used instead of Rhodamine R6 in the staining procedure for the sham-operated group. The staining results in A1 and A2 are consistent, showing a weak positive signal in neurons in the CA1 region; however, no positive signal was observed in A3.

[0102] B1 is a fluorescence image of the CA1 region of the hippocampus stained with Rhodamine R6 after 2 days of ischemia / reperfusion. B2 is a light micrograph of the Rhodamine R6 fluorescence signal converted into DAB / peroxidase reaction products in the 2-day ischemia / reperfusion group. B3 shows the 2-day ischemia / reperfusion group treated with 0.1M phosphate buffer instead of Rhodamine R6. The staining results in B1 and B2 are similar, with a large number of positive neurons visible in the CA1 region; however, no positive neurons were observed in B3.

[0103] Combination Figure 6 Electron microscopy revealed that although structures such as the nucleus, endoplasmic reticulum, and mitochondria were visible in the cytoplasm of neurons in the sham-operated group, no obvious peroxidase / DAB reaction products were observed.

[0104] exist Figure 6 In the diagram, A represents pyramidal neurons in the CA1 region of the hippocampus from the sham-operated group; B is a magnified view of a portion of diagram A.

[0105] In the control group, normal structures such as the nucleus, endoplasmic reticulum, and mitochondria were visible in the cytoplasm of neurons, but no obvious peroxidase / DAB reaction products were observed.

[0106] Combination Figure 7 As can be seen, electron microscopy observations show that, compared with Figure 6 Compared to the sham-operated group, the number of DAB / peroxidase products in the CA1 region neurons of ischemic / reperfused rats was significantly increased. These products were mainly distributed in autophagosome-like structures in the neuronal cytoplasm, damaged endoplasmic reticulum and mitochondria, and various vesicular structures. This result is consistent with confocal microscopy findings. Figure 3 The results are consistent with those shown.

[0107] exist Figure 7 In the diagram, A represents pyramidal neurons damaged in the CA1 region of the hippocampus after 2 days of ischemia / reperfusion; compared with the control group ( Figure 6 Compared to the previous year, DAB / peroxidase products were significantly increased in neurons of the CA1 region of ischemic / reperfused rats. These products were mainly distributed in autophagosome-like structures in the neuronal cytoplasm (indicated by the arrow in Figure B) and in the damaged endoplasmic reticulum (C). Figure 3 The positive markers are indicated by the angular shape and mitochondria (pointed to by the single arrow in Figure C), as well as various vesicular structures (D). However, no positive markers are seen in normal mitochondria (indicated by the double arrows in Figure C).

[0108] Combination Figure 8 Electron microscopy revealed that severely damaged neurons were filled with positively labeled vesicle-like structures. However, if Rhodamine R6 was omitted from the reaction procedure, although the cytoplasm of the damaged neurons was filled with vesicles, almost no positive labels were visible.

[0109] exist Figure 8 In Figure A, CA1 neurons are labeled with the product of the DAB / peroxidase reaction after Rhodamine R6 fluorescence conversion. These cells are filled with positively labeled vesicle-like structures. Figure B shows that Rhodamine R6 was omitted in the reaction step. Although the cytoplasm of the damaged neurons is filled with vesicles, almost no positive structures can be seen.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting neuronal death using a combination of fluorescence and electron microscopy, characterized in that, The method for detecting neuronal death using fluorescence and electron microscopy coupling includes the following steps: Step 1: A rat model of transient global cerebral ischemia was created using Pulsinelli's four-vessel occlusion method, and samples were taken from the model animals. Step 2: The specimens obtained in Step 1 were stained with monochromatic fluorescence. Then, the Rhodamine R6 fluorescent label in the sections was converted into 3,3'-diaminobenzidine precipitate. The sections were then prepared as electron microscopy specimens, and the experimental results were measured.

2. The method for detecting neuronal death by coupled fluorescence and electron microscopy according to claim 1, characterized in that, Step one describes the creation of a rat model of global cerebral ischemia using Pulsinelli's four-vessel occlusion method. The sampling and processing of the model animals includes the following steps: S1. Anesthetize rats by intraperitoneal injection of 10% chloral hydrate at a ratio of 0.4 ml / 100 g. Fix the rats in a supine position, wipe the skin in the middle of the rat's neck with an alcohol swab and trim the rat hair, and disinfect with povidone-iodine. S2. Make a 2cm incision in the middle of the rat's neck, and separate the sternocleidomastoid muscle outward along the medial border of the sternocleidomastoid muscle. Separate the fascia around the common carotid artery and free a section of blood vessel for threading. Then treat the contralateral artery in the same way. After completion, suture the skin and disinfect with povidone-iodine. S3. Fix the rat in a prone position, disinfect it using the method described in S1, make a 1cm incision in the middle of the back of the neck, use blunt forceps to separate the paravertebral muscles from the middle to expose the first cervical vertebral lamina, and then move it outward and upward to expose the pterygoid foramina on both sides. S4. Insert the electrocoagulator into the wing hole to perform electrocoagulation. After electrocoagulation, suture the muscle and skin, wipe with iodine for disinfection, and put it back in the cage. S5. Wait 24 hours, remove the rat, fix it in a supine position, expose the common carotid arteries on both sides using the method described in S2, clamp them with arterial clamps, and then release the fixation. The disappearance of the righting reflex and corneal reflex in the rat within 15-30 seconds proves that the whole brain ischemia was successful. Discard rats that have convulsions during the process. S6. After clamping both common carotid arteries for 13 minutes, remove the clamps to restore blood flow; then suture the skin, disinfect with povidone-iodine, and return the animal to its cage for continued feeding. S7. 1 to 14 days after ischemia-reperfusion, the experimental animals were anesthetized with 10% chloral hydrate, the thoracic cavity was opened to expose the heart, and the perfusion pump was used to operate through the ascending aorta. The blood was first flushed with physiological saline, and then 4% paraformaldehyde was used for perfusion fixation. S8. After perfusion, remove the animal's brain and place it in 4% paraformaldehyde for 4-6 hours. Then, preserve the brain tissue in 5% sucrose. S9. Use a vibratory slicer to cut brain tissue into sections 30-50 μm thick, wash three times with 0.1 M phosphate buffer for 5-15 minutes each time, and store at 4°C.

3. The method for detecting neuronal death using fluorescence and electron microscopy coupled according to claim 1, characterized in that, The method for detecting neuronal death using fluorescence and electron microscopy coupling as described in step two includes the following steps: S1. Collect the slices into a 24-well plate and immerse them in a 0.5-3% hydrogen peroxide solution. React on a shaker for 10-30 minutes. S2. Add a potassium permanganate aqueous solution with a concentration of 0.06~0.25% and a pH of 4.5~7.5 to pretreat the slices for 5~15 minutes; S3. Briefly rinse the pretreated slides with distilled water, then rinse three times with 0.1M PB buffer for 5-10 minutes each time. S4. Add Rhodamine R6 working solution and incubate at room temperature in the dark for 5 min to 2 h. The negative control group is replaced with 0.1 M phosphate buffer instead of Rhodamine R6. S5. Wash the incubated slices three times with 0.1M PB buffer, each time for 5-10 minutes; S6. Some sections were stained according to the FluoroJade B (FJ-B) staining procedure to confirm neuronal death, and some sections were stained with 4',6-diamidinyl-2-phenylindole to confirm changes in cell nuclear morphology. After staining, the sections were picked up and placed on a glass slide for observation and photography using a fluorescence microscope. S7. After observation under a fluorescence microscope, the Rhodamine R6 stained sections were placed back into the 24-well plate and incubated at room temperature for 0.5 to 1.0 hours in 0.1M phosphate buffer containing 4-10% normal goat serum and 0.05-0.1% polyethylene glycol octylphenyl ether. S8. Discard the solution from S7, add mouse monoclonal anti-rhodamine antibody, and incubate overnight at 4°C. S9. Wash the sections three times with 0.1M phosphate buffer, add goat anti-mouse IgG H&L (Biotin) secondary antibody, and incubate at room temperature for 1-2 hours; Wash three times with S10 and 0.1M phosphate buffer for 5-15 minutes each time, then add the avidin-biotin-peroxidase complex and react at room temperature for 0.5-1.5 hours. S11. Wash the reacted sections three times with 0.1M phosphate buffer for 5-15 minutes each time, and then wash with 50mM Tris buffer at pH 7.4 for 5-15 minutes. S12. Add 50 mM Tris buffer containing 0.001~0.05% 3,3'-diaminobenzidine and react at room temperature for 5~15 minutes. S13. Discard the solution in S12, add 50mM Tris buffer containing 0.005~0.03% hydrogen peroxide and 0.001~0.05% 3,3'-diaminobenzidine, and react at room temperature for 5~15 minutes. S14. First, wash the slides with 50mM Tris buffer at pH 7.4 for 5-15 minutes, then wash the slides three times with 0.1M phosphate buffer for 5-15 minutes each time. Finally, add 0.1M phosphate buffer at pH 4.5-7.4 containing 0.75-1.00% osmium tetroxide and react at 4°C for 0.5-2 hours. S15. Dehydrate the sections in a gradient manner according to concentrations of 50%, 70%, 80%, 90%, 95%, 100% I, and 100% II. Then, add propylene oxide to replace ethanol for 5-15 minutes and repeat 1-2 times. S16. Weigh Epon812 embedding agent, dodecenylsuccinic anhydride, methylnonylacetaldehyde, and 2,4,6-tris(dimethylaminomethyl)phenol, prepare the embedding solution according to the ordinary electron microscope preparation method, and add the section to a 1:1 volume ratio mixture of propylene oxide and embedding solution, and incubate overnight at room temperature. S17. Place the slices into a plastic embedding container, add embedding solution, react at 40°C for 1 day, and then react at 60°C for 2 days to allow the embedding solution to fully polymerize. S18. Using an ultramicrotome, the embedded specimens were prepared into ultrathin sections with a thickness of 80-120 nm and collected on a copper grid. The samples from the ischemia / reperfusion group stained with 3,3'-diaminobenzidine were observed directly under an electron microscope after ultrathin sectioning. Ultrathin sections of S19, the sham-operated group, and the negative control group (with Rhodamine R6 omitted) were stained with 2.0–6.0% uranyl acetate for 3 minutes, then washed three times with distilled water for 2 minutes each time. Next, the ultrathin sections were stained with 0.1–0.5% lead citrate for 2.5 minutes, then washed three times with distilled water for 2 minutes each time. After drying, they were used for electron microscopy.

4. The method for detecting neuronal death using fluorescence and electron microscopy coupled according to claim 3, characterized in that, The Rhodamine R6 is prepared as a stock solution with a concentration of 1.0 mg / ml using ethanol or dimethyl sulfoxide and stored at -20°C. Before use, it is prepared as a working solution with buffer solution at a concentration of 500~5000×.

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