Dyeing marking method for caenorhabditis elegans

Abstract

The invention relates to a dyeing marking method for caenorhabditis elegans, buffer solutions used in the method are all at low temperature, and centrifugal operation is carried out at low temperature. According to the dyeing marking method of the caenorhabditis elegans, the loss rate of the caenorhabditis elegans sample can be reduced to be less than 30% from the original more than 50%, the dyeing time is greatly shortened, the dyeing efficiency is greatly improved, and meanwhile, the subcutaneous lipid droplet distribution of the caenorhabditis elegans can be clearly and accurately displayed; and finally obtaining a high-definition caenorhabditis elegans dyeing picture which is neatly arranged, equivalent in dyeing degree, free of bubbles, background impurities or color interference.

Description

Technical Field

[0001] The present invention relates to the technical field of lipid droplet detection, in particular to a staining and labeling method for Caenorhabditis elegans. Background Art

[0002] Caenorhabditis elegans (C. elegans) is a harmless, self-sustaining nematode. Small, with adults measuring only 1 mm long, it has a simple structure and a transparent body, making it a model organism for molecular and developmental biology research.

[0003] Oil Red O staining is an effective method for detecting fat storage in the nematode Caenorhabditis elegans. Oil Red O is a lipid-soluble azo dye with strong lipid solubility, specifically binding to neutral triglycerides, lipids, and lipoproteins within tissues and cells. When tissue or cell sections are placed in an Oil Red O staining solution, the dye leaves the solution and dissolves in the lipids within the tissue or cells. Because Oil Red O is more soluble in lipids than in organic solvents, the dye transfers from the solution to the lipids. Once bound to lipids, the lipids appear red or orange, facilitating the observation and analysis of lipid status within cells or tissues.

[0004] However, with the existing technology, there are still many problems with Oil Red O staining of C. elegans: (1) The nematode loss rate is high: the nematode loss rate is at least greater than 50% from collection to final mounting; (2) The staining time is long and the efficiency is low: the Oil Red O staining time mentioned in the literature is as short as 2 hours and as long as overnight; (3) The display of subcutaneous lipid droplets in nematodes is not clear enough: the subcutaneous lipid droplets of adults are more abundant than those of larvae, but many experiments involving Oil Red O staining of adults only observed the lipid droplet content in the intestine, while the subcutaneous lipid droplets were very unclear; (4) The final image is not neat and beautiful: the images of nematodes after Oil Red O staining reported in many literatures are arranged in a messy manner and do not meet the standards for publishing high-level papers. Summary of the Invention

[0005] Problems to be solved by the invention

[0006] In response to the above-mentioned problems existing in the prior art, the present invention aims to provide a more efficient and accurate nematode oil red staining method, which can minimize the loss rate for experiments with small sample quantities, and at the same time provide a more aesthetically pleasing mounting and output method for those who need to publish high-level articles.

[0007] Solutions for solving problems

[0008] The invention provides a dyeing and marking method for Caenorhabditis elegans. The buffer solution used in the method is all low-temperature, and the centrifugation operation is all performed at low temperature.

[0009] Preferably, the low temperature is 1 to 10°C;

[0010] More preferably, the low temperature is 4°C.

[0011] Preferably, the centrifugal force of the centrifugation is 1500 to 3000 g;

[0012] More preferably, the centrifugal force of the centrifugation is 2000 g.

[0013] Preferably, the method comprises the following steps:

[0014] (1) Collect the nematodes using buffer A and wash them by centrifugation;

[0015] (2) Tissue fixation using paraformaldehyde and buffer B;

[0016] (3) Prepare Oil Red O solution and isopropyl alcohol solution respectively;

[0017] (4) After fixation, remove the fixative by centrifugation using buffer C;

[0018] (5) removing water from the isopropanol solution and centrifuging the solution, and then staining with the Oil Red O solution;

[0019] (6) Use buffer D and buffer C to remove the stain by centrifugation and then mount the nematodes.

[0020] Preferably, the buffer A in step (1) is a weak ionization buffer specifically for Caenorhabditis elegans, and more preferably, the buffer A in step (1) is M9 buffer;

[0021] Preferably, the buffer B in step (2) is MRWB buffer, more preferably, the MRWB buffer contains 160 mM KCl, 40 mM NaCl, 14 mM Na2EGTA, 1 mM spermidine hydrochloride, 0.4 mM spermine, 30 mM Na-PIPES, and 0.2% β-mercaptoethanol;

[0022] Preferably, the buffer C in step (4) is PBS buffer;

[0023] Preferably, the buffer D in step (6) is PBST buffer, more preferably, the PBST buffer is 1x PBS buffer containing 1% Triton X-100.

[0024] Preferably, the paraformaldehyde in step (2) is a 4% paraformaldehyde solution;

[0025] Preferably, the Oil Red O solution in step (3) is a 60% Oil Red O solution;

[0026] Preferably, the isopropanol solution in step (3) is a 60% isopropanol solution.

[0027] Preferably, the Oil Red O solution in step (3) is prepared in a large-sized centrifuge tube;

[0028] More preferably, the volume ratio of the Oil Red O solution in step (3) to the large-sized centrifuge tube is 1:5 to 1:30;

[0029] Further preferably, the volume ratio of the Oil Red O solution to the large-size centrifuge tube in step (3) is 1:15 to 1:25;

[0030] Preferably, the dyeing time in step (5) is 1 to 2 hours.

[0031] More preferably, the staining time in step (5) is 1 hour.

[0032] Preferably, the cover sheet used in the mounting in step (6) is a vertical cover sheet.

[0033] Preferably, the mounting in step (6) comprises the following steps:

[0034] (6a) arranging nematodes in a culture medium;

[0035] (6b) Add droplet 1 of buffer A onto the agar plate;

[0036] (6c) placing the arranged nematodes into the droplet I described in step (6b);

[0037] (6d) Add a droplet II of buffer A onto one side of the coverslip;

[0038] (6e) After the droplet I in step (6b) has completely evaporated, the droplet II in step (6d) is directed toward the nematodes arranged in step (6c), and the cover is placed vertically to complete the mounting.

[0039] Preferably, the buffer A is a weak ionization buffer specifically for Caenorhabditis elegans, more preferably, the buffer A is M9 buffer;

[0040] Preferably, the agar slice in step (6b) is a tablet made of agarose solution.

[0041] Preferably, the volume of the droplet I in step (6b) is greater than the volume of the droplet II in step (6d);

[0042] Preferably, the volume of the droplet I in step (6b) is 3 to 4 μL;

[0043] Preferably, the volume of the droplet II in step (6d) is 1 to 2 μL.

[0044] Effects of the Invention

[0045] The staining and labeling method of the present invention can reduce the loss rate of nematode samples from more than 50% to less than 30%, greatly shortening the staining time and staining efficiency while also being able to clearly and accurately display the distribution of subcutaneous lipid droplets in the nematodes, ultimately obtaining a high-definition staining image of the nematodes that is neatly arranged, has a comparable staining degree, is free of bubbles, and is free of background impurities or color interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Oil red staining and average optical density statistics.

[0047] Figure 2 Figure 2 shows the staining and labeling of Caenorhabditis elegans using room temperature buffer.

[0048] Figure 3 elegans staining using cryo-buffer.

[0049] Figure 4 This is a staining labeling example for Caenorhabditis elegans using small-format microcentrifuge tubes.

[0050] Figure 5 Marking conditions for staining Caenorhabditis elegans using large-format centrifuge tubes.

[0051] Figure 6 elegans staining using the traditional coverslipping method.

[0052] Figure 7 Figure 2. Staining and labeling of Caenorhabditis elegans using a vertical coverslipping method. DETAILED DESCRIPTION

[0053] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0054] As used herein, the term "M9 buffer" refers to a weak ionization buffer specifically for Caenorhabditis elegans, which can maintain the cell osmotic pressure of nematodes.

[0055] In certain embodiments, the preparation method of the M9 buffer comprises: dissolving 3 g of potassium dihydrogen phosphate, 6 g of disodium hydrogen phosphate, and 5 g of sodium chloride in 1 L of single-distilled water, sterilizing by high pressure and returning to room temperature, adding 1 mL of 1 M magnesium sulfate, stirring evenly, and storing at 4°C.

[0056] As used herein, the term "NGM" refers to Nematode Growth Medium, ie, nematode growth medium.

[0057] In certain embodiments, the NGM preparation method comprises: dissolving 16 g agar powder, 2 g tryptone, and 2.4 g sodium chloride in 800 mL of single-distilled water, sterilizing by autoclaving, equilibrating the culture medium temperature to 55-65° C., and then adding 800 uL of 1 M magnesium sulfate, 5 mg / mL cholesterol, and 800 uL of 1 M calcium chloride.

[0058] As used herein, the term "MRWB buffer" refers to a buffer containing 160 mM KCl, 40 mM NaCl, 14 mM Na2EGTA, 1 mM spermidine hydrochloride, 0.4 mM spermine, 30 mM Na-PIPES, and 0.2% β-mercaptoethanol.

[0059] As used herein, the term "PBS buffer" refers to phosphate buffered saline.

[0060] In certain embodiments, the PBS buffer is Labshark: 130114004.

[0061] As used herein, the term "PBST buffer" refers to 1x PBS buffer containing 1% Triton X-100.

[0062] The invention provides a dyeing and marking method for Caenorhabditis elegans. The buffer used in the method is all low-temperature, and the centrifugation operation is all performed at low temperature.

[0063] In certain embodiments, the low temperature is 1-10°C.

[0064] In certain embodiments, the low temperature is 1°C, or 1.5°C, or 2°C, or 2.5°C, or 3°C, or 3.5°C, or 4°C, or 4.5°C, or 5°C, or 5.5°C, or 6°C, or 6.5°C, or 7°C, or 7.5°C, or 8°C, or 8.5°C, or 9°C, or 9.5°C, or 10°C.

[0065] In certain embodiments, the low temperature is 4°C.

[0066] In certain embodiments, the centrifugal force of the centrifugation is 1500-3000 g.

[0067] In certain embodiments, the centrifugal force of the centrifugation is 1500g, or 1600g, or 1700g, or 1800g, or 1900g, or 2000g, or 2100g, or 2200g, or 2300g, or 2400g, or 2500g, or 2600g, or 2700g, or 2800g, or 2900g, or 3000g.

[0068] In certain embodiments, the centrifugal force of the centrifugation is 2000 g.

[0069] In certain embodiments, the method comprises the steps of:

[0070] (1) Collect the nematodes using buffer A and wash them by centrifugation;

[0071] (2) Tissue fixation using paraformaldehyde and buffer B;

[0072] (3) Prepare Oil Red O solution and isopropyl alcohol solution respectively;

[0073] (4) After fixation, remove the fixative by centrifugation using buffer C;

[0074] (5) removing water from the isopropanol solution and centrifuging the solution, and then staining with the Oil Red O solution;

[0075] (6) Use buffer D and buffer C to remove the stain by centrifugation and then mount the nematodes.

[0076] In certain embodiments, the buffer A in step (1) is a weak ionization buffer specifically for Caenorhabditis elegans.

[0077] In certain embodiments, the buffer A in step (1) is M9 buffer.

[0078] In certain embodiments, the step (1) comprises collecting the nematodes using buffer A, centrifuging and allowing the mixture to stand, discarding the supernatant, and resuspending the nematodes using buffer A.

[0079] In some embodiments, the step (1) is to collect the nematodes using buffer A, centrifuge at 2000g and 4°C for 30s, let it stand on ice for 30s, discard the supernatant, resuspend the nematodes using buffer A, and repeat the centrifugation and washing steps two to three times.

[0080] In certain embodiments, after the last centrifugation in step (1), the standing time is extended to 1 min.

[0081] In certain embodiments, the paraformaldehyde in step (2) is a 4% paraformaldehyde solution.

[0082] In certain embodiments, the buffer B in step (2) is MRWB buffer.

[0083] In certain embodiments, the buffer B in step (2) is 2x MRWB buffer.

[0084] In certain embodiments, the MRWB buffer contains 160 mM KCl, 40 mM NaCl, 14 mM Na2EGTA, 1 mM spermidine hydrochloride, 0.4 mM spermine, 30 mM Na-PIPES, and 0.2% β-mercaptoethanol.

[0085] In certain embodiments, the volume ratio of the paraformaldehyde to buffer B in step (2) is 2:1 to 1:2.

[0086] In certain embodiments, the volume ratio of the paraformaldehyde to buffer B in step (2) is 2:1, or 1.9:1, or 1.8:1, or 1.7:1, or 1.6:1, or 1.5:1, or 1.4:1, or 1.3:1, or 1.2:1, or 1.1:1, or 1:1, or 1:1.1, or 1:1.2, or 1:1.3, or 1:1.4, or 1:1.5, or 1:1.6, or 1:1.7, or 1:1.8, or 1:1.9, or 1:2.

[0087] In certain embodiments, the volume ratio of the paraformaldehyde to buffer B in step (2) is 1:1.

[0088] In certain embodiments, step (2) comprises resuspending the nematodes using paraformaldehyde and buffer B, and rotating.

[0089] In certain embodiments, the rotation speed is 10-25 rpm.

[0090] In some embodiments, the rotation speed is 10rpm, 11rpm, 12rpm, 13rpm, 14rpm, 15rpm, 16rpm, 17rpm, 18rpm, 19rpm, 20rpm, 21rpm, 22rpm, 23rpm, 24rpm, or 25rpm.

[0091] In certain embodiments, the rotating speed is 18 rpm.

[0092] In certain embodiments, the rotation time is 40 to 60 minutes.

[0093] In some embodiments, the rotation time is 40 min, or 41 min, or 42 min, or 43 min, or 44 min, or 45 min, or 46 min, or 47 min, or 48 min, or 49 min, or 50 min, or 51 min, or 52 min, or 53 min, or 54 min, or 55 min, or 56 min, or 57 min, or 58 min, or 59 min, or 60 min.

[0094] In certain embodiments, the rotating time is 50 minutes.

[0095] In certain embodiments, during the rotation, the liquid in the centrifuge tube flows in an upside-down state.

[0096] In certain embodiments, the Oil Red O solution in step (3) is a 60% Oil Red O solution.

[0097] In certain embodiments, the isopropanol solution in step (3) is a 60% isopropanol solution.

[0098] In certain embodiments, the Oil Red O solution in step (3) is prepared in a large-sized centrifuge tube.

[0099] In certain embodiments, the volume ratio of the Oil Red O solution in step (3) to the large-sized centrifuge tube is 1:5 to 1:30.

[0100] In certain embodiments, the volume ratio of the Oil Red O solution in step (3) to the large-format centrifuge tube is 1:5, or 1:6, or 1:7, or 1:8, or 1:9, or 1:10, or 1:11, or 1:12, or 1:13, or 1:14, or 1:15, or 1:16, or 1:17, or 1:18, or 1:19, or 1:20, or 1:21, or 1:22, or 1:23, or 1:24, or 1:25, or 1:26, or 1:27, or 1:28, or 1:29, or 1:30.

[0101] In certain embodiments, the volume ratio of the Oil Red O solution in step (3) to the large-sized centrifuge tube is 1:15 to 1:25.

[0102] In certain embodiments, the staining time in step (5) is 1 to 2 hours.

[0103] In certain embodiments, the staining time in step (5) is 1 h, or 1.1 h, or 1.2 h, or 1.3 h, or 1.4 h, or 1.5 h, or 1.6 h, or 1.7 h, or 1.8 h, or 1.9 h, or 2 h.

[0104] In certain embodiments, the staining time in step (5) is 1 hour.

[0105] In certain embodiments, the preparation of the Oil Red O solution in step (3) is to dilute the saturated isopropanol Oil Red O solution to 60% by volume using deionized double distilled water in a large-sized centrifuge tube protected from light, and then rotate and filter.

[0106] In certain embodiments, the rotation speed is 10-25 rpm.

[0107] In some embodiments, the rotation speed is 10rpm, 11rpm, 12rpm, 13rpm, 14rpm, 15rpm, 16rpm, 17rpm, 18rpm, 19rpm, 20rpm, 21rpm, 22rpm, 23rpm, 24rpm, or 25rpm.

[0108] In certain embodiments, the rotating speed is 18 rpm.

[0109] In certain embodiments, the rotation time is 50 to 70 minutes.

[0110] In some embodiments, the rotation time is 50 min, or 51 min, or 52 min, or 53 min, or 54 min, or 55 min, or 56 min, or 57 min, or 58 min, or 59 min, or 60 min, or 61 min, or 62 min, or 63 min, or 64 min, or 65 min, or 66 min, or 67 min, or 68 min, or 69 min, or 70 min.

[0111] In certain embodiments, the rotating time is 60 minutes.

[0112] In certain embodiments, the push filtration membrane is an organic phase filter membrane.

[0113] In certain embodiments, the pore size of the push filter membrane is 0.45 μm.

[0114] In certain embodiments, the preparation of the isopropanol solution in step (3) is performed by diluting the saturated isopropanol solution to 60% by volume using deionized double-distilled water in a large-sized centrifuge tube protected from light.

[0115] In certain embodiments, the volume ratio of the deionized double-distilled water to the saturated isopropanol solution is 2:3.

[0116] In certain embodiments, the buffer C in step (4) is PBS buffer.

[0117] In certain embodiments, the buffer C in step (4) is 1x PBS buffer.

[0118] In certain embodiments, step (4) comprises adding buffer C to the solution obtained by fixation in step (2), centrifuging the solution, allowing the solution to stand, discarding the supernatant, and resuspending the nematodes in buffer C.

[0119] In certain embodiments, step (4) comprises adding buffer C to the solution fixed in step (2), centrifuging at 2000 g and 4° C. for 30 s, letting it stand on ice for 30 s, discarding the supernatant, resuspending the nematodes in buffer C, and repeating the centrifugation and washing steps once.

[0120] In certain embodiments, the dehydration in step (5) comprises adding the isopropanol solution prepared in step (3) to the product of step (4) and rotating the mixture.

[0121] In certain embodiments, the rotation speed is 10-25 rpm.

[0122] In some embodiments, the rotation speed is 10rpm, 11rpm, 12rpm, 13rpm, 14rpm, 15rpm, 16rpm, 17rpm, 18rpm, 19rpm, 20rpm, 21rpm, 22rpm, 23rpm, 24rpm, or 25rpm.

[0123] In certain embodiments, the rotating speed is 18 rpm.

[0124] In certain embodiments, the rotation time is 1 to 10 minutes.

[0125] In certain embodiments, the rotation time is 1 min, or 2 min, or 3 min, or 4 min, or 5 min, or 6 min, or 7 min, or 8 min, or 9 min, or 10 min.

[0126] In certain embodiments, the rotation time is 5 minutes.

[0127] In certain embodiments, the staining with the Oil Red O solution in step (5) comprises centrifuging the dehydrated solution and then allowing it to stand, discarding the isopropanol, adding the Oil Red O solution prepared in step (3), and rotating.

[0128] In certain embodiments, the rotation speed is 10-25 rpm.

[0129] In some embodiments, the rotation speed is 10rpm, 11rpm, 12rpm, 13rpm, 14rpm, 15rpm, 16rpm, 17rpm, 18rpm, 19rpm, 20rpm, 21rpm, 22rpm, 23rpm, 24rpm, or 25rpm.

[0130] In certain embodiments, the rotating speed is 18 rpm.

[0131] In certain embodiments, the rotation time is 1 to 2 hours.

[0132] In certain embodiments, the rotation time is 1 h, or 1.1 h, or 1.2 h, or 1.3 h, or 1.4 h, or 1.5 h, or 1.6 h, or 1.7 h, or 1.8 h, or 1.9 h, or 2 h.

[0133] In certain embodiments, the rotating time is 1 hour.

[0134] In certain embodiments, the Oil Red O solution in step (5) is prepared immediately before use.

[0135] In certain embodiments, the buffer D in step (6) is PBST buffer.

[0136] In certain embodiments, the PBST buffer is 1× PBS buffer containing 1% Triton X-100.

[0137] In certain embodiments, step (6) includes centrifuging and then standing, discarding the oil red, resuspending the nematodes in buffer D, rotating, and then washing the nematodes in buffer C before mounting.

[0138] In some embodiments, step (6) includes centrifugation at 2000 g and 4° C. for 30 s, standing on ice for 30 s, discarding the oil red, resuspending the nematodes in buffer D, repeating the centrifugation and washing steps once, then washing the nematodes in buffer C, centrifuging at 2000 g and 4° C. for 30 s, standing on ice for 30 s, discarding the supernatant, and mounting the nematodes.

[0139] In certain embodiments, the cover sheet used in the mounting in step (6) is a vertical cover sheet.

[0140] In some embodiments, the mounting in step (6) comprises the following steps:

[0141] (6a) arranging nematodes in a culture medium;

[0142] (6b) Add droplet 1 of buffer A onto the agar plate;

[0143] (6c) placing the arranged nematodes into the droplet I described in step (6b);

[0144] (6d) Add a droplet II of buffer A onto one side of the coverslip;

[0145] (6e) After the droplet I in step (6b) has completely evaporated, the droplet II in step (6d) is directed toward the nematodes arranged in step (6c), and the cover is placed vertically to complete the mounting.

[0146] In certain embodiments, the culture medium is NGM.

[0147] In certain embodiments, the buffer A is a weak ionization buffer specifically for Caenorhabditis elegans.

[0148] In certain embodiments, the buffer A is M9 buffer.

[0149] In certain embodiments, the agar slice in step (6b) is a tablet made of agarose solution.

[0150] In certain embodiments, the agarose solution is a 2.5% agarose solution.

[0151] In certain embodiments, the volume of the droplet I in step (6b) is greater than the volume of the droplet II in step (6d).

[0152] In certain embodiments, the volume of the droplet I in step (6b) is 3 to 4 μL.

[0153] In certain embodiments, the volume of the droplet II in step (6d) is 1 to 2 μL.

[0154] In certain embodiments, the method further comprises the steps of:

[0155] (7) Use a high-resolution microscope in bright field mode to take pictures.

[0156] Example 1: Caenorhabditis elegans staining and labeling

[0157] 1. Collect nematodes: Use 4°C 1x M9 buffer to wash the nematodes from the NGM into a 2 mL centrifuge tube. If the sample size is small, select ≥50 nematodes and place them into 1 mL of 4°C 1x M9 buffer.

[0158] 2. Wash the nematodes: Centrifuge at 2000g for 30 seconds at 4°C and place on ice for 30 seconds. At this point, the nematodes should have settled and the bacterial suspension should be suspended in the supernatant. Discard the supernatant and reserve approximately 300 μL of the supernatant. Resuspend the nematodes in 700 μL of 4°C 1x M9 buffer. Repeat the same centrifugation and standing method for two to three times.

[0159] 3. Tissue Fixation: After the final centrifugation of the washed nematodes, extend the rest time to 1 min. Discard as much of the supernatant as possible (retain approximately 100 μL) and add 300 μL of 4% paraformaldehyde (bioshark, BL539A) and 300 μL of 2x MRWB buffer to resuspend the nematode pellet. Then, rotate the centrifuge tube at room temperature (approximately 20-25°C) on a rotator at 18 rpm for 50 min. During the rotation, be sure to adjust the angle to ensure that the liquid in the centrifuge tube is flowing upside down.

[0160] 4. Prepare 60% Oil Red O solution: Wrap a sterile 50 mL centrifuge tube with tin foil to protect it from light. Then, dilute the saturated isopropanol Oil Red O solution (Sigma, O1391) to 60% volume fraction with deionized double distilled water (ddH2O) in the centrifuge tube. Rotate the centrifuge tube at 18 rpm for 1 h at room temperature. After 1 h, filter the solution into another sterile centrifuge tube using a sterile syringe and a 0.45 μm filter membrane for the organic phase. This completes the preparation of 60% Oil Red O (2 ml).

[0161] 5. Prepare 60% isopropanol solution: Take a sterile 50 mL centrifuge tube, wrap it with tin foil to protect it from light, and then prepare a 60% volume fraction of isopropanol solution in a ratio of ddH2O:isopropanol = 2:3. Tighten the cap and store at room temperature.

[0162] 6. Rinse the fixative: After 50 minutes of tissue fixation, add 1 mL of 4°C 1x PBS buffer to 600 μL of fixative. Centrifuge at 2000g for 30 seconds at 4°C and place on ice for 30 seconds. At this point, the nematodes have basically settled. Discard the supernatant and keep about 300 μL. Resuspend the nematodes in 700 μL of 4°C 1x PBS buffer. Continue using the same centrifugation and standing method to wash the nematodes once more.

[0163] 7. Dehydration of nematodes: After the final wash, discard as much supernatant as possible (retain about 100 μL), then add 1 mL of 60% isopropanol solution, and then rotate the centrifuge tube on a room temperature rotator at 18 rpm for 5 minutes.

[0164] 8. Oil Red O staining: After dehydration, centrifuge the tube at 2000g for 30 seconds at 4°C and place on ice for 30 seconds until the nematodes have basically settled. Discard the supernatant (isopropanol) and add 1 mL of prepared 60% Oil Red O solution. Then, rotate the centrifuge tube at 18 rpm on a room temperature rotator for 1-2 hours to stain.

[0165] 9. Stain Removal: Centrifuge at 2000g for 30 seconds at 4°C and place on ice for 30 seconds until the nematodes have essentially settled. Discard the Oil Red and resuspend the nematodes in 1 mL of 4°C 1x PBST buffer (1x PBS buffer containing 1% Triton X-100). Rotate the tube rack at 18 rpm for 1 minute at room temperature to wash away the stain. Using the same centrifugation and standing conditions, discard the supernatant again (retaining approximately 200 μL). Add 1 mL of 4°C 1x PBS and rinse the worms one final time (inverting the tube about eight times). Centrifuge and let stand, then discard the supernatant, reducing the volume to approximately 200 μL.

[0166] 10. Nematode mounting: Prepare several NGM plates that have not been inoculated with E. coli. Use a low-absorption pipette to transfer the nematode suspensions from different experimental groups to the NGM. After the liquid evaporates, pick nematodes of similar length or age and arrange them in a clean area of the NGM. Then, using two slides, press 2.5% agarose solution (melted at 95°C in advance) into an agar plate and drop about 3-4 μL of room temperature 1x M9 buffer on it. Pick up the nematodes that have been arranged in advance in the NGM and place them into the M9 droplet at once. Further adjust the nematode arrangement before the liquid evaporates. After the liquid evaporates completely, take a clean coverslip and drop about 1-2 μL of room temperature 1x M9 buffer on one side. Then, turn the cover slip facing down and let the M9 drop fall vertically above the arranged worms. The nematode mounting is complete.

[0167] 11. Photography: Within 1 hour after mounting, place the mounted nematodes on an upright or inverted high-resolution microscope, select bright field mode for photography, and adjust the background white balance to appropriate parameters (try to avoid a dark background that makes it difficult to distinguish the staining details). The same data can be used for photography in the same group of experiments. Oil red staining and average optical density statistics were performed on the untreated wild-type nematodes (WT) and the nematodes fed with high cholesterol (High cho.). The results were Figure 1 As shown, after nematodes were fed with high cholesterol, neutral lipids accumulated in their bodies, thereby increasing the coloration of oil red staining, and the average optical density value AOD analyzed by imageJ showed statistical differences.

[0168] Comparative Example 1: Using room temperature and low-speed centrifugation to stain and label Caenorhabditis elegans

[0169] The whole centrifugation process was performed at room temperature and 1000 g, and the remaining steps were the same as those in Example 1.

[0170] The results showed that at the end of the final dyeing, 100 nematodes were selected for dyeing in Comparative Example 1, and only 20-30 samples remained. However, 100 nematodes were selected for dyeing in Example 1, and more than 80 nematodes remained. The remaining nematode amount in Example 1 was significantly increased, and the loss rate was low.

[0171] Comparative Example 2: Caenorhabditis elegans staining using room temperature buffer

[0172] The nematodes were collected and washed using room temperature M9 buffer, and all washing steps were performed using room temperature PBS buffer. The remaining steps were the same as in Example 1.

[0173] like Figure 2 As shown in Figure 2, using room temperature buffer and room temperature centrifugation, it can be seen that the distribution of lipid droplets in the head, intestine, and tail is not obvious; Figure 3 As shown, using cryobuffer and cold centrifugation conditions, lipid droplets are clearly distributed in the head, gut, and tail. The cryobuffer, combined with increased centrifugal force and cold centrifugation conditions, reduces nematode loss. A cold environment reduces the fluidity of lipid droplets and membranes, helping to preserve and stabilize the lipid droplets. Therefore, nematodes treated with cryobuffer exhibit a clearer and more accurate visualization of the distribution and content of neutral lipid droplets.

[0174] Comparative Example 3: Staining and labeling of Caenorhabditis elegans using small-sized centrifuge tubes

[0175] A 5 mL centrifuge tube was used for the dissolution step in preparing the 60% Oil Red O dye solution, and the remaining steps were the same as in Example 1.

[0176] like Figure 4 As shown, the oil red O dye solution dissolved in a small volume centrifuge tube was used for staining, and the oil red staining was lighter; Figure 5 As shown, staining with Oil Red O dye dissolved in a large-volume centrifuge tube shows clear and bright Oil Red coloring. Rotating the large-volume centrifuge tube allows more room for the 60% Oil Red O to flow, greatly increasing its solubility and dissolution efficiency, which lays the foundation for the subsequent staining effect.

[0177] Comparative Example 4: Staining and labeling of Caenorhabditis elegans using traditional coverslipping

[0178] The stained nematodes were directly placed on the agarose slide and placed in a swaying motion, and then covered with a coverslip using an inclined pressing method. The remaining steps were the same as in Example 1.

[0179] like Figure 6 As shown in Figure 2, various bubbles can often be seen in the field of view of nematodes mounted using traditional methods, and the arrangement of nematodes is not uniform; Figure 7As shown, tilting the coverslip causes unidirectional liquid flow, dispersing the nematodes. Using a vertical pressing method for mounting ensures that the nematodes are always neatly arranged, with a flat surface around the worms and no air bubbles. This method significantly reduces the air bubble problem that occurs when tilting the coverslip, improving the final slide appearance. Pre-positioning the worms on NGM solid medium also improves mounting efficiency.

[0180] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A dyeing and labeling method for Caenorhabditis elegans, characterized in that: The buffers used in the method are all low-temperature, and the centrifugation operations are all performed at low temperatures.

2. The method according to claim 1, characterized in that The low temperature is 1 to 10°C; Preferably, the low temperature is 4°C.

3. The method according to claim 1, characterized in that The centrifugal force of the centrifugation is 1500-3000g; Preferably, the centrifugal force of the centrifugation is 2000g.

4. The method according to claim 1, wherein The method comprises the following steps: (1) Collect the nematodes using buffer A and wash them by centrifugation; (2) Tissue fixation using paraformaldehyde and buffer B; (3) Prepare Oil Red O solution and isopropyl alcohol solution respectively; (4) After fixation, remove the fixative by centrifugation using buffer C; (5) removing water from the isopropanol solution and centrifuging the solution, and then staining with the Oil Red O solution; (6) Use buffer D and buffer C to remove the stain by centrifugation and then mount the nematodes.

5. The method according to claim 4, characterized in that The buffer A in step (1) is a weak ionization buffer specifically for Caenorhabditis elegans. Preferably, the buffer A in step (1) is M9 buffer; Preferably, the buffer B in step (2) is MRWB buffer, more preferably, the MRWB buffer contains 160mM KCl, 40mM NaCl, 14mM Na2EGTA, 1mM spermidine hydrochloride, 0.4mM spermine, 30mM Na-PIPES, and 0.2% β-mercaptoethanol; Preferably, the buffer C in step (4) is PBS buffer; Preferably, the buffer D in step (6) is PBST buffer, more preferably, the PBST buffer is 1x PBS buffer containing 1% Triton X-100.

6. The method according to claim 4, characterized in that The paraformaldehyde in step (2) is a 4% paraformaldehyde solution; Preferably, the Oil Red O solution in step (3) is a 60% Oil Red O solution; Preferably, the isopropanol solution in step (3) is a 60% isopropanol solution.

7. The method according to claim 4, characterized in that The Oil Red O solution in step (3) is prepared in a large-sized centrifuge tube; Preferably, the volume ratio of the Oil Red O solution in step (3) to the large-sized centrifuge tube is 1:5 to 1:30; More preferably, the volume ratio of the Oil Red O solution in step (3) to the large-sized centrifuge tube is 1:15 to 1:25; Preferably, the dyeing time in step (5) is 1 to 2 hours. More preferably, the staining time in step (5) is 1 hour.

8. The method according to claim 4, characterized in that The cover sheet method used in the mounting in step (6) is a vertical cover sheet.

9. The method according to claim 8, characterized in that The mounting in step (6) comprises the following steps: (6a) arranging nematodes in a culture medium; (6b) Add droplet 1 of buffer A onto the agar plate; (6c) placing the arranged nematodes into the droplet I described in step (6b); (6d) Add a droplet II of buffer A onto one side of the coverslip; (6e) After the droplet I in step (6b) has completely evaporated, the droplet II in step (6d) is directed toward the nematodes arranged in step (6c), and the cover is placed vertically to complete the mounting.

10. The method according to claim 9, characterized in that The buffer A is a weak ionization buffer specially used for Caenorhabditis elegans, preferably, the buffer A is M9 buffer; Preferably, the agar slice in step (6b) is a tablet made of agarose solution. Preferably, the volume of the droplet I in step (6b) is greater than the volume of the droplet II in step (6d); Preferably, the volume of the droplet I in step (6b) is 3 to 4 μL; Preferably, the volume of the droplet II in step (6d) is 1 to 2 μL.

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