Method for detecting mitochondrial membrane potential of gill or hepatopancreas of crustacean

Abstract

This invention discloses a method for detecting mitochondrial membrane potential in the gills or hepatopancreas of crustaceans, belonging to the field of mitochondrial membrane potential detection technology. This invention, by addressing the structural and physiological differences between gill and hepatopancreas tissues in crustaceans, formulates differentiated incubation strategies: for gill tissue, a "live pre-equilibration-short-time staining-rapid slide preparation" method is used, strictly controlling the JC-1 incubation time to within 10 minutes to effectively avoid probe toxicity damage to mitochondria; for hepatopancreas tissue, in vivo incubation is extended to 1 hour to ensure sufficient dye penetration. Under the condition of uniformly using 2 μM JC-1 and 20 μmol / L CCCP as positive controls, this method significantly improves the tissue penetration and signal uniformity of the dye, solving problems such as uneven staining and weak signal caused by exoskeleton barriers and high lipid content, providing reliable technical support for aquatic health assessment and toxicology research.

Description

Technical Field

[0001] This invention relates to the field of mitochondrial membrane potential detection technology, and in particular to a method for detecting the mitochondrial membrane potential of the gills or hepatopancreas of crustaceans. Background Technology

[0002] Mitochondrial membrane potential (ΔΨm) is a key parameter reflecting the functional state of mitochondria, and its detection typically relies on fluorescent probes such as JC-1. This probe forms red-fluorescent aggregates or green-fluorescent monomers within the cell based on the membrane potential, and the membrane potential level is assessed by changes in the fluorescence signal ratio. Currently, in fish or mammalian tissues, routine JC-1 detection often employs in vitro tissue immersion staining or direct incubation methods. These methods are simple to operate and are common technical solutions for ΔΨm detection in cells and tissues.

[0003] However, when the aforementioned general methods are applied to the gills and hepatopancreas tissues of crustaceans (such as shrimp), significant limitations are revealed. First, due to the dense exoskeleton structure of crustacean tissues and the chitinous covering on the surface of gill tissues, dye molecules have difficulty penetrating effectively, resulting in weak staining signals or severely uneven distribution. Second, there are significant differences between different tissue types: gill tissues are rich in blood flow and have a relatively loose structure, while hepatopancreas tissues have a high lipid content and a dense structure. The different adsorption and retention capacities of the two tissues lead to significant differences in staining kinetics, making it difficult to obtain ideal results through uniform incubation time and concentration conditions. In addition, the physiological state of crustacean tissues is extremely unstable after being removed from the body. The long incubation or mechanical manipulation required by conventional methods can easily introduce additional stress damage, which may cause non-physiological collapse of mitochondrial membrane potential before detection, resulting in false positive signals or loss of true signals. The root cause of these defects is that existing technical solutions have failed to be specifically optimized for the unique anatomical structures of crustaceans (such as the exoskeleton barrier) and the physiological differences of different tissues (gills and hepatopancreas), and also lack reliable positive controls to verify the effectiveness of the detection system itself, ultimately resulting in low reliability and poor repeatability of the detection results.

[0004] Therefore, there is an urgent need to establish a mitochondrial membrane potential detection method that can be optimized for the characteristics of shrimp gills or hepatopancreas tissues, ensuring effective dye penetration and signal intensity while minimizing damage during in vitro manipulation. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting the mitochondrial membrane potential of the gills or hepatopancreas of crustaceans, thereby addressing the problems existing in the prior art. This invention, by taking into account the structural and physiological differences between the gills and hepatopancreas tissues of crustaceans, formulates differentiated incubation strategies. This effectively avoids damage to mitochondria caused by probe toxicity and significantly improves the tissue penetration and signal uniformity of the dye, solving problems such as uneven staining and weak signal caused by the exoskeleton barrier and high lipid content.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for detecting the membrane potential of mitochondria in the gills or hepatopancreas of crustaceans. The method includes incubating healthy, vigorous crustaceans in a NaCl solution containing JC-1 under a light-protected environment, removing them, immediately taking gill or hepatopancreas tissue for slide preparation, and rapidly acquiring dual-channel fluorescence images. The membrane potential of the mitochondria in the gills or hepatopancreas of the crustaceans is determined based on the ratio of the average fluorescence intensity of the TRITC / TRITC channel to the FITC / GFP channel in the same field of view.

[0008] When the object to be detected is the gill mitochondrial membrane potential, the incubation time is 5-10 minutes;

[0009] When the target being tested is the mitochondrial membrane potential of the liver and pancreas, the incubation time is 60 minutes.

[0010] Furthermore, the concentration of JC-1 is 2 μM.

[0011] Furthermore, the mass concentration of the NaCl solution is 6.5–7‰.

[0012] Furthermore, the temperature throughout the entire testing process was 28°C.

[0013] Furthermore, the crustaceans are crustaceans that have undergone pretreatment by soaking in NaCl solution.

[0014] Furthermore, the pretreatment includes immersing the crustacean in a NaCl solution with a mass concentration of 6.5–7‰ to equilibrate it.

[0015] When the target being tested is the gill mitochondrial membrane potential, the immersion equilibration time is 30 min; when the target being tested is the hepatopancreatic mitochondrial membrane potential, the immersion equilibration time is 60 min.

[0016] Furthermore, the crustaceans include shrimp.

[0017] Furthermore, the shrimp species include the Pacific white shrimp.

[0018] Furthermore, the detection method uses the healthy, vigorous crustaceans as a positive control, which are first immersed in a NaCl solution containing 20 μmol / L carbonyl cyanochlorophenylhydrazone.

[0019] Furthermore, when the target being tested is the gill mitochondrial membrane potential, the soaking time is 30 minutes; when the target being tested is the hepatopancreatic mitochondrial membrane potential, the soaking time is 60 minutes.

[0020] The present invention discloses the following technical effects:

[0021] This invention addresses the structural and physiological differences between gill and hepatopancreatic tissues in crustaceans by developing differentiated incubation strategies: for gill tissue, a "live pre-equilibration-short staining-rapid slide preparation" method is employed, strictly controlling the JC-1 incubation time to within 10 minutes to effectively avoid probe toxicity-induced mitochondrial damage; for hepatopancreatic tissue, incubation is extended to 1 hour to ensure sufficient dye penetration. Under the condition of using 2 μM JC-1 and 20 μmol / L CCCP as positive controls, this method significantly improves dye tissue penetration and signal uniformity, resolving issues such as uneven staining and weak signal caused by the exoskeleton barrier and high lipid content.

[0022] This method is simple to operate and highly reproducible. The entire process involves in vivo immersion staining, minimizing tissue stress and non-physiological membrane potential collapse caused by in vitro manipulation, effectively reducing the risk of false positives or false negatives. By introducing CCCP chemical depolarization verification, the relationship between red / green fluorescence signals and membrane potential changes is clearly demonstrated, enhancing the reliability and persuasiveness of the detection results. This strategy has well-defined parameters and controllable conditions, and can be extended to mitochondrial function studies in other crustaceans, providing reliable technical support for aquatic health assessment and toxicology research. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of the fluorescence microscope showing the JC-1 staining of the gills of Litopenaeus vannamei; where A is the green channel image of the detection group; B is the red channel image of the detection group; C is the green channel image of the CCCP-treated group; and D is the red channel image of the CCCP-treated group.

[0025] Figure 2This is a schematic diagram of the fluorescence microscope showing JC-1 staining of the hepatopancreas of Litopenaeus vannamei; where A is the green channel diagram of the detection group; B is the red channel diagram of the detection group; C is the green channel diagram of the CCCP-treated group; and D is the red channel diagram of the CCCP-treated group.

[0026] Figure 3 The red / green fluorescence intensity ratio was used to detect the mitochondrial membrane potential of gill tissue in Litopenaeus vannamei. Data are expressed as mean ± standard deviation (n=3 in the detection group and n=3 in the CCCP group). Statistical analysis was performed using an independent samples t-test. *p<0.05 indicates a significant difference between the two groups.

[0027] Figure 4 The red / green fluorescence intensity ratio was used to detect the mitochondrial membrane potential of Litopenaeus vannamei hepatopancreatic tissue. Data are expressed as mean ± standard deviation (n=3 in the detection group and n=3 in the CCCP group). Statistical analysis was performed using an independent samples t-test. ****p<0.001 indicates a significant difference between the two groups. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Example

[0034] 1. Preparation before the experiment

[0035] Experimental animals: Healthy and vigorous Litopenaeus vannamei shrimp, weighing 3–4 g each, were selected. Before the experiment, they were temporarily housed in a NaCl solution at 28°C and a concentration of 6.5–7‰ for at least 24 hours.

[0036] Main reagents and preparation:

[0037] Physiological saline: simulates the seawater environment, with a salinity precisely configured to 6.5–7 wt‰.

[0038] JC-1 Stock Solution and Working Solution: High-purity JC-1 fluorescent probe (CAS No.: 3520-43-2) was used. The stock solution was prepared with DMSO and further diluted with the above-mentioned physiological saline to ensure that the concentration of JC-1 in the final injection working solution was 2 μM.

[0039] CCCP positive control solution: The mitochondrial uncoupling agent CCCP (carbonyl cyanochlorophenylhydrazone, CAS No.: 555-60-2) was prepared with physiological saline to ensure that the concentration of CCCP in the final injection working solution was 20 μmol / L.

[0040] Instruments and equipment: fluorescence microscope (equipped with FITC / GFP channels (green channel) for JC-1 monomers and TRITC / TRITC channels (red channel) for aggregates), dissecting instruments, light-proof incubation containers, etc.

[0041] Environmental control: The entire experimental process, including solution preparation, tissue incubation, and dissection, was conducted under light-protected conditions to prevent the fluorescent dye from photoquenching. The experimental ambient temperature was kept constant at 28℃.

[0042] 2. Detection of mitochondrial membrane potential in gill tissue

[0043] This method takes advantage of the rapid staining of gill tissue and its sensitivity to JC-1 toxicity by employing a strategy of "in vivo pre-equilibration - short-time staining - rapid slide preparation".

[0044] 2.1 Detection group (normal detection ΔΨm)

[0045] a. Live equilibration: Take a live whiteleg shrimp and place it in a light-proof container containing 4 L of NaCl solution with a mass concentration of 6.5–7‰. Immerse it at a constant temperature of 28℃ for 30 min to equilibrate.

[0046] b. JC-1 staining: Add an appropriate amount of pre-prepared JC-1 stock solution directly to the above soaking system to achieve a final working concentration of 2 μM. Gently mix and continue incubation in the dark. The incubation time should be strictly controlled within 5–10 min to avoid damage to the gill mitochondria caused by the autotoxicity of JC-1 after 10 min.

[0047] c. Sample Preparation and Image Acquisition: Immediately remove the shrimp from the solution and quickly dissect them to obtain gill tissue. Place the gill tissue on a glass slide, cover it with a coverslip, and quickly observe it under a fluorescence microscope. Using the same exposure time and gain parameters, acquire fluorescence images in the green channel (JC-1 monomer, excitation / emission approximately 514 / 529 nm) and the red channel (JC-1 aggregate, excitation / emission approximately 585 / 590 nm), and save them as raw TIFF files.

[0048] 2.2 CCCP treatment group (positive control, used to verify the measurability of ΔΨm)

[0049] a. CCCP pre-incubation: In another set of experiments, CCCP stock solution was added to 4 L of NaCl solution with a mass concentration of 6.5–7‰ to bring the final working concentration to 20 μmol / L. Live shrimp were placed in this solution and incubated at 28°C in the dark for 30 min to fully uncouple the mitochondria and dissipate the membrane potential.

[0050] b. JC-1 staining: After CCCP incubation, without changing the solution, add JC-1 stock solution directly to the system to make the final concentration 2 μM, and continue incubation in the dark for no more than 10 min.

[0051] c. Sample preparation and image acquisition: Same as step 2.1c, immediately remove the gill tissue for slide preparation and quickly acquire dual-channel fluorescence images.

[0052] 3. Detection of mitochondrial membrane potential in liver and pancreatic tissues

[0053] This method takes advantage of the dense structure of hepatocellular and pancreatic tissues and the slow penetration of dyes by employing a strategy of "extended in vivo incubation".

[0054] 3.1 Test Group (Normal ΔΨm)

[0055] a. Live equilibration: Take a live whiteleg shrimp and place it in a light-proof container containing 4 L of suitable brine with a salinity of 6.5–7‰. Soak it at a constant temperature of 28℃ for 60 minutes to equilibrate.

[0056] b. JC-1 staining: Add JC-1 stock solution to the soaking system to achieve a final working concentration of 2 μM. Continue incubation at 28°C in the dark for 1 hour to ensure sufficient time for the dye to penetrate and enter the hepatopancreatic cells.

[0057] c. Sample preparation and image acquisition: Immediately after incubation, the hepatopancreas tissue was dissected, slides were prepared, and red and green dual-channel fluorescence images were quickly acquired under a fluorescence microscope. All parameters were consistent with those used for gill tissue analysis.

[0058] 3.2 CCCP treatment group (positive control)

[0059] a. CCCP pre-incubation: First, add CCCP stock solution to 4 L of NaCl solution with a mass concentration of 6.5–7‰ to bring the final working concentration to 20 μmol / L. Place live shrimp in this solution and incubate for 1 h at 28°C in the dark.

[0060] b. JC-1 staining: Subsequently, without changing the solution, add JC-1 stock solution directly to a final concentration of 2 μM, and continue incubation in the dark for 1 hour.

[0061] c. Sample preparation and image acquisition: Same as step 3.1c, immediately dissect and sample, prepare slides and complete dual-channel image acquisition.

[0062] 4. Analysis and Verification

[0063] The acquired fluorescence images are then analyzed. Typically, ImageJ or similar image analysis software is used to calculate the ratio of the average fluorescence intensity of the red channel to the green channel in the same field of view (red / green ratio). This ratio is positively correlated with the mitochondrial membrane potential level.

[0064] 5. Results

[0065] Figure 1 This is a schematic diagram of a fluorescence microscope showing the staining of the gills of Litopenaeus vannamei (whiteleg shrimp) with JC-1 staining. Figure 1 A is the green channel diagram of the detection group. It can be seen that the overall background green signal is extremely weak, with only weak scattered points, indicating that mitochondria mainly exist in the form of JC-1 aggregation. Figure 1 B is the red channel image of the detection group, which shows that the gill tissue exhibits strong red fluorescence (JC-1 aggregates), indicating that the mitochondria are in a high membrane potential (polarized state). Figure 1 C is the green channel diagram of the CCCP-treated group, which shows a significant enhancement of green fluorescence, indicating an increase in JC-1 monomers and reflecting the loss of mitochondrial membrane potential; Figure 1D is the red channel image of the CCCP-treated group, showing a significant reduction in red fluorescence with only a small amount of residual punctate fluorescence remaining. This indicates that the method of the present invention can obtain a stable JC-1 signal in gill tissue, and the red / green fluorescence signal distribution changes significantly after CCCP depolarization treatment, proving that the method can accurately reflect changes in mitochondrial membrane potential.

[0066] Figure 2 This is a schematic diagram of the fluorescence microscopy of the hepatopancreas stained with JC-1 in Litopenaeus vannamei, consistent with the fluorescence results of JC-1 staining in the gills. The green channel image of the detection group shows a very weak overall green signal. Figure 2 The A-value indicates that JC-1 mainly exists as aggregates; the red channel image shows obvious red fluorescence in the hepatopancreatic tissue. Figure 2 The B signal indicates that the mitochondria are in a high membrane potential state. In the CCCP-treated group, the green channel image shows significantly enhanced green fluorescence, while in the red channel image, red fluorescence is almost completely absent, with only a few scattered signals remaining. Figure 2 (C and D). This demonstrates that the immersion staining method of the present invention can obtain reliable JC-1 fluorescence signals in hepatopancreatic tissues, and that the fluorescence distribution changes significantly with regularity after CCCP treatment, making it an effective indicator of mitochondrial membrane potential changes.

[0067] The red / green fluorescence ratios of the detection group and the CCCP treatment group were statistically compared (t-test), and the results are as follows: Figure 3 and Figure 4 As shown: Figure 3 The results showed the ratio of JC-1 red / green fluorescence intensity in the gill tissue of Litopenaeus vannamei in the test group and the CCCP-treated group. The mean red / green fluorescence ratio of the CCCP-treated group (0.2754) was significantly lower than that of the test group (2.5870), a reduction of approximately 89%. Figure 4 The results show the JC-1 red / green fluorescence intensity ratio of hepatopancreatic tissue from Litopenaeus vannamei in the detection group and the CCCP-treated group. The mean red / green fluorescence ratio of the CCCP-treated group (0.2082) was significantly lower than that of the detection group (2.0223), a reduction of approximately 90%. This indicates that in the detection of gills and hepatopancreatic tissue, the ratio in the CCCP group was significantly lower than that in the detection group, demonstrating that the detection method of this invention has high sensitivity to changes in ΔΨm.

[0068] The above embodiments demonstrate that the detection method of the present invention can effectively and sensitively respond to changes in ΔΨm. For gill tissue, the method of the present invention can obtain a stronger and more uniform red fluorescence signal with low background fluorescence, avoiding false negatives (signal quenching) or false positives (toxic damage) caused by over-incubation; for hepatopancreatic tissue, prolonged incubation ensures sufficient dye penetration, and the signal intensity is significantly better than that of short-time incubation protocols.

[0069] In summary, this invention, by setting differentiated and optimized JC-1 incubation times (gills ≤10 min, hepatopancreas 60 min) for two tissues with significant structural and physiological differences in shrimp, namely gills and hepatopancreas, and by standardizing the key dye concentration (2 μM) and positive control conditions (20 μmol / L CCCP), ensures signal intensity and uniformity while minimizing tissue stress and damage caused by in vitro manipulation and probe toxicity, thereby achieving reliable and accurate detection of mitochondrial membrane potential in key shrimp tissues.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for detecting the mitochondrial membrane potential of the gills or hepatopancreas of crustaceans, characterized in that, The process includes incubating healthy, vigorous crustaceans in a NaCl solution containing JC-1 under a light-protected environment, removing them, immediately taking gill or hepatopancreatic tissue for slide preparation, and rapidly acquiring dual-channel fluorescence images. The membrane potential of the mitochondria in the gills or hepatopancreas of the crustaceans is determined based on the ratio of the average fluorescence intensity of the TRITC / TRITC channel to the FITC / GFP channel in the same field of view. The concentration of JC-1 was 2 μM; The NaCl solution has a mass concentration of 6.5–7‰; The crustaceans mentioned are crustaceans that have undergone pretreatment by soaking in NaCl solution; The pretreatment includes immersing the crustaceans in a NaCl solution with a mass concentration of 6.5–7‰ to achieve equilibration; When the object being tested is the gill mitochondrial membrane potential, the immersion equilibration time is 30 min; when the object being tested is the hepatopancreatic mitochondrial membrane potential, the immersion equilibration time is 60 min. When the object to be detected is the gill mitochondrial membrane potential, the incubation time is 5-10 minutes; When the target being tested is the mitochondrial membrane potential of the liver and pancreas, the incubation time is 60 minutes. The temperature throughout the testing process was 28℃.

2. The detection method according to claim 1, characterized in that, The crustaceans mentioned include shrimp.

3. The detection method according to claim 2, characterized in that, The shrimp species mentioned include the whiteleg shrimp (Litopenaeus vannamei).

4. The detection method according to claim 1, characterized in that, The detection method uses healthy, vigorous crustaceans as a positive control, which are first immersed in a NaCl solution containing 20 μmol / L carbonyl cyanochlorophenylhydrazone.

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