A rapid preparation method for cytoskeleton experimental samples

Abstract

The present invention discloses a rapid preparation method for cytoskeleton experimental samples. Tear the inner epidermis of an onion bulb, add phosphate buffer solution, and after the inner epidermis of the onion bulb sinks, perform microwave treatment; suck out the phosphate buffer solution, add an extraction agent, a 1wt% Triton X-100 solution to cover the onion sample, and perform microwave treatment; suck out the extraction agent, soak and wash with M-buffer solution, and perform microwave treatment; suck out the M-buffer solution, add a fixing solution, a 3wt% glutaraldehyde solution to cover the onion sample, and perform microwave treatment; suck out the fixing solution, soak and wash with phosphate buffer solution, and perform microwave treatment; suck out the phosphate buffer solution, add a 0.2wt% Coomassie Brilliant Blue R250 staining solution and perform microwave treatment; rinse with distilled water to remove the floating solution, cover with a cover glass and observe. By using microwave treatment in the present invention, the entire experimental process can be shortened to be completed within 2 hours. After treatment, the cytoskeleton structure is clear and complete, and the overall operation is simple, which can be popularized and used in undergraduate experiments.

Description

Technical Field

[0001] The present invention belongs to the technical field of cytoskeleton experiment samples, and particularly relates to a rapid preparation method for cytoskeleton experiment samples. Background Art

[0002] The cytoskeleton is a fibrous reticular three-dimensional structure observable inside eukaryotic cells, and mainly includes microtubules, microfilaments and intermediate filaments. The cytoskeleton plays an important role in maintaining cell morphology, cell movement, intracellular material transport and other aspects. Therefore, the cytoskeleton is one of the most active fields in modern cell biology research and an important content of cell biology experiments.

[0003] In recent years, scholars have continuously explored and optimized cytoskeleton experiments. Tan Juan, Qian Xinping, Fu Xiuqin, Tong Shukun, Feng Zhenyue, etc. have respectively explored the conditions of cytoskeleton observation experiments and optimized steps such as the concentration of extraction reagent, extraction time, fixative, fixation time and staining time. However, the classical Coomassie brilliant blue staining method is still used. This method mostly uses plant cells as experimental materials. After treating cells with Triton X-100, cytoskeletal proteins are retained, then the actin is stabilized with M buffer, then fixed with glutaraldehyde, and finally stained with Coomassie brilliant blue-R250. After each step, it is rinsed with PBS. The whole process takes 3 - 4 hours. Although the experimental operation is simple, the time consumption is relatively long. Summary of the Invention

[0004] The technical problem solved by the present invention is to provide a rapid preparation method for cytoskeleton experiment samples, which can greatly improve the time utilization efficiency.

[0005] The present invention adopts the following technical solutions to solve the above technical problems. A rapid preparation method for cytoskeleton experiment samples is characterized in that the specific steps are as follows:

[0006] Step S1, Tear the inner epidermis of an onion bulb and place it in a cube plastic box, add 0.2 mol / L phosphate buffer solution. After the inner epidermis of the onion bulb sinks, place it in a microwave oven and heat it at 30 - 40 °C for 2 min;

[0007] Step S2, Suck out the phosphate buffer solution, add 1 wt% Triton X-100 solution as the extraction agent to cover the onion sample, place it in a microwave oven and heat it at 30 - 40 °C for 5 min;

[0008] Step S3, suck out the extraction agent, soak and wash with M-buffer three times, and heat-treat each time in a microwave oven at 30 - 40 °C for 2 min, where the M-buffer consists of 50 mmol / L imidazole, 50 mmol / L potassium chloride, 0.5 mmol / L magnesium chloride, 1 mmol / L EGTA, 0.1 mmol / L ethylenediaminetetraacetic acid, and 1 mmol / L β-mercaptoethanol;

[0009] Step S4, suck out the M-buffer, add a fixing solution of 3 wt% glutaraldehyde solution to cover the onion sample, and heat-treat in a microwave oven at 30 - 40 °C for 5 min;

[0010] Step S5, suck out the fixing solution, soak and wash with phosphate buffer three times, and heat-treat each time in a microwave oven at 30 - 40 °C for 2 min;

[0011] Step S6, suck out the phosphate buffer, add 0.2 wt% Coomassie Brilliant Blue R250 staining solution and heat-treat in a microwave oven at 30 - 40 °C for 3 min;

[0012] Step S7, rinse 1 - 2 times with distilled water to remove the floating liquid, cover with a cover glass and observe.

[0013] Further defined, the extraction agent of 1 wt% Triton X-100 solution in Step S2 is prepared from M-buffer.

[0014] Further defined, the fixing solution of 3 wt% glutaraldehyde solution in Step S4 is prepared from phosphate buffer.

[0015] Further defined, the solvent of the 0.2 wt% Coomassie Brilliant Blue R250 staining solution in Step S6 consists of the following solutions by volume percentage: 46.5% methanol, 7% glacial acetic acid, and 46.5% distilled water.

[0016] Further defined, the sample in each step needs to be placed in a beaker filled with water when being processed in the microwave oven, and the water in the beaker should be replaced in time after each treatment to avoid the water temperature in the beaker exceeding 40 °C.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: After microwave treatment, the whole experimental process of the present invention can be completed within 2 hours. After treatment, the cytoskeleton structure is clear and complete, and the overall operation is simple, which can be popularized and used in undergraduate experiments. At the same time, the requirements for exploratory experiments in undergraduate experimental teaching are getting higher and higher, but usually, exploratory experiments take a long time and the experimental scheme design is complex. However, this experiment has a simple operation, which allows students to independently explore the optimal conditions from multiple aspects such as extraction time, fixation time, and treatment temperature, so as to cultivate students' independent experimental ability. Description of the Drawings

[0018] Figure 1 It is a diagram showing the effect of extraction microwave treatment time on the cytoskeleton;

[0019] Figure 2 It is a diagram showing the effect of fixed microwave treatment time on the cytoskeleton;

[0020] Figure 3 It is a diagram showing the effect of treatment temperature on the cytoskeleton. Detailed implementation manners

[0021] The above content of the present invention will be further described in detail below through examples, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Examples

[0022] 1 Experimental materials and methods

[0023] 1.1.1 Materials: Onion epidermis

[0024] 1.1.2 Reagents

[0025] (1) M-buffer: 50 mmol / L imidazole, 50 mmol / L potassium chloride, 0.5 mmol / L magnesium chloride, 1 mmol / L EGTA, 0.1 mmol / L ethylenediaminetetraacetic acid, and 1 mmol / L β-mercaptoethanol.

[0026] (2) Phosphate buffer solution (PBS): 6 mmol / L, pH = 6.8.

[0027] (3) 1.0 wt% Triton X-100 solution: Prepared with M-buffer.

[0028] (4) 3.0 wt% glutaraldehyde solution: Prepared with phosphate buffer solution.

[0029] (5) 0.2 wt% Coomassie Brilliant Blue R250 staining solution: Its solvent consists of the following solutions by volume percentage: 46.5% methanol, 7% glacial acetic acid, and 46.5% distilled water.

[0030] 1.1.3 Instruments

[0031] Microwave oven: Midea M1-L213B

[0032] Microscope: Nikon 100

[0033] 1.2 Conventional treatment method for cytoskeleton experiment

[0034] 1.2.1 Sampling and rinsing: Tear off the inner epidermis of the onion (1 cm square) and place it in PBS (pH = 6.8) buffer solution for 8 minutes.

[0035] 1.2.2 Extraction: Aspirate the PBS buffer solution, and extract the inner epidermis of the onion with 1 wt% Triton X-100 extractant for 25 min to remove miscellaneous proteins.

[0036] 1.2.3 Washing: Aspirate the extractant, and soak and wash with M-buffer solution three times, 10 min each time.

[0037] 1.2.4 Fixation: Fix with 3 wt% glutaraldehyde solution at room temperature for 45 min.

[0038] 1.2.5 Re-washing: Aspirate the fixative, and soak and wash with PBS buffer solution three times, 10 min each time.

[0039] 1.2.6 Staining: Stain with 0.2 wt% Coomassie Brilliant Blue R250 staining solution for 25 min.

[0040] 1.2.7 Slide preparation: Rinse with distilled water, cover with a coverslip, and make a temporary mount for observation.

[0041] 1.3 Improvement of the experimental method

[0042] The improved method is that the treatment method for each experimental operation step is changed to be treated with medium-low heat in a microwave oven, and the operation steps remain unchanged. At the same time, the treatment time of the key steps is optimized. The specific operations are as follows:

[0043] (1) Tear off the inner epidermis of the onion bulb (1 cm square) and place it in a 2.5 cm side length cube plastic box. Add 0.2 mol / L phosphate buffer solution (PBS). After the inner epidermis of the onion bulb sinks, place it in a microwave oven and heat-treat it with medium-low heat for 2 min.

[0044] (2) Extraction: Aspirate the buffer solution, add 1 wt% Triton X-100 solution of the extractant to cover the onion sample, and place it in a microwave oven and heat-treat it with medium-low heat.

[0045] (3) Washing: Aspirate the extractant, soak and wash with M-buffer solution three times, and each time place it in a microwave oven and heat-treat it with medium-low heat for 2 min.

[0046] (4) Fixation: Aspirate the M-buffer solution, add 3 wt% glutaraldehyde solution of the fixative to cover the onion sample, and place it in a microwave oven and heat-treat it with medium-low heat.

[0047] (5) Re-washing: Aspirate the fixative, soak and wash with PBS buffer solution three times, and each time place it in a microwave oven and heat-treat it with medium-low heat for 2 min.

[0048] (6) Staining: Aspirate the PBS buffer solution, add 0.2 wt% Coomassie Brilliant Blue R250 staining solution and place it in a microwave oven and heat-treat it with medium-low heat for 3 min.

[0049] (7) Slide preparation and microscopic examination: Rinse with distilled water 1 - 2 times to remove the floating liquid, cover with a cover slip and observe.

[0050] During the microwave treatment process, to keep the liquid temperature constant, the sample box can be placed in a 1L beaker filled with water, making the sample box float on the water surface. At the same time, place another 1L beaker filled with water in the microwave oven to assist in heat absorption. After each microwave treatment, the water in the beaker should be replaced in a timely manner.

[0051] In the classic Coomassie brilliant blue staining method, it mainly relies on the specific binding of Coomassie brilliant blue R250 to proteins. To exclude the influence of proteins in the cell membrane and cytoplasm, it is necessary to dissolve and extract the proteins in the cell membrane and cytoplasm with Triton X - 100, and then fix with glutaraldehyde for easy observation. Therefore, in this experiment, the extraction time and fixation time are used as the optimized parameters, and the influence of temperature on the results is also considered.

[0052] 1.3.1 Optimization of the microwave treatment time for extraction

[0053] Using 1wt% Triton X - 100 as the extraction agent, during the extraction process, the microwave treatment times are set to 3 min, 5 min, and 8 min respectively to explore the influence of the microwave treatment time for extraction on the cytoskeleton.

[0054] 1.3.2 Optimization of the microwave treatment time for fixation

[0055] Using 3wt% glutaraldehyde as the fixative, during the fixation process, the microwave treatment times are set to 3 min, 5 min, and 8 min respectively to explore the influence of the microwave treatment time for fixation on the cytoskeleton.

[0056] 1.3.3 Optimization of the treatment temperature

[0057] Keep the temperature of the water in the beaker at 30 - 40 °C, 50 - 60 °C, and 70 - 80 °C respectively to explore the influence of the treatment temperature on the cytoskeleton.

[0058] 2 Experimental results

[0059] 2.1 Influence of the microwave treatment time for extraction on the cytoskeleton

[0060] As Figure 1As shown in the figure, three extraction times were set in this study. When the extraction time was 3 min, there were many colored lumps in the cells, making the observation background of the cytoskeleton unclear. This might be because the extraction time was too short to completely extract the miscellaneous proteins in the cytoskeleton. When the extraction time was 8 min, the arrangement of the cytoskeleton was discontinuous and rather blurred, probably due to the overlong extraction time causing cytoskeleton depolymerization. When the extraction time was 5 min, the observation effect of the cytoskeleton was the best. Therefore, a 5-min extraction time was used in the subsequent experiments of this study.

[0061] 2.2 Effect of fixation microwave treatment time on the cytoskeleton

[0062] As Figure 2 shown in the figure, three fixation times were set in this study. When the cells were treated with the fixative for 5 min and 8 min, the cytoskeleton was relatively clear and the observation effect was relatively ideal. Although the cytoskeleton was also relatively clear when fixed for 3 min, the arrangement of the cytoskeleton was relatively loose and the observation effect was not good. Ideal observation results could also be obtained when fixed for 8 min. Considering saving time, a 5-min fixation time was used in the subsequent experiments of this study.

[0063] 2.3 Effect of treatment temperature on the cytoskeleton

[0064] As Figure 3 shown in the figure, two temperatures were set in this study. The cytoskeleton could be clearly shown during the treatment at 30 - 40 °C, and the observation results were relatively ideal. While during the treatment at 50 - 60 °C, only relatively thick microstructures could be preserved, most of the microfilaments were broken and discontinuous, and the observation results were not ideal. Therefore, the treatment temperature of 30 - 40 °C was used in this study.

[0065] 3 Experimental conclusions

[0066] By comparing the experimental results, the rapid preparation method of the cytoskeleton by the microwave method is summarized as follows:

[0067] (1) Tear off the inner epidermis of the onion bulb (1 cm square) and place it in a 2.5-cm side length cube plastic box. Add 0.2 mol / L PBS buffer solution. After the inner epidermis of the onion bulb sinks, place it in a microwave oven and heat-treat it at 30 - 40 °C for 2 min.

[0068] (2) Extraction: Aspirate the PBS buffer solution, add 1 wt% Triton X-100 solution as the extractant to cover the onion epidermis sample, and place it in a microwave oven and heat-treat it at 30 - 40 °C for 5 min.

[0069] [[ID=3']](3) Washing: Aspirate the extractant, soak and wash it 3 times with M-buffer solution, and each time place it in a microwave oven and heat-treat it at 30 - 40 °C for 2 min.

[0070] (4)Fixation: Aspirate the M-buffer solution, cover the onion epidermal sample with a fixing solution of 3 wt% glutaraldehyde solution, and place it in a microwave oven for heat treatment at 30 - 40 °C for 5 min.

[0071] (5)Washing again: Aspirate the fixing solution, soak and wash with PBS buffer solution three times, and each time place it in a microwave oven for heat treatment at 30 - 40 °C for 2 min.

[0072] (6)Staining: Aspirate the PBS buffer solution, add 0.2 wt% Coomassie Brilliant Blue R250 staining solution and place it in a microwave oven for heat treatment at 30 - 40 °C for 3 min.

[0073] (7)Preparing slides for microscopy: Rinse with distilled water 1 - 2 times to remove the floating liquid, cover with a coverslip, and observe with a 40× objective lens.

[0074] To maintain a stable temperature, the sample should be placed in a beaker filled with water when being processed in the microwave oven. After each treatment, the water in the beaker should be replaced in a timely manner to prevent the temperature of the water in the beaker from exceeding 40 °C.

[0075] 4 Conclusion

[0076] Microwave is an electromagnetic wave with a frequency in the range of 300 MHz - 300 GHz, which can cause bipolar molecules such as water molecules and proteins to vibrate at high speed by 180°. This high-speed movement between and within molecules can not only make the tissue generate heat, but also promote the diffusion of reagents in the tissue and the chemical reaction rate, shortening the time for the reagent to act. When the sample is irradiated with microwaves, the polar molecules of the reagent and the sample will change their orientations with the changing electromagnetic field, causing the reagent molecules and the sample molecules to collide and rub against each other. The surfaces of both the reagent and the sample are continuously updated, and the surface of the sample continuously contacts new reagent molecules, promoting the acceleration of the chemical reaction between the reagent and the sample. The reason for using low-power microwave radiation in the experiment is on the one hand to avoid too rapid an increase in the temperature of the sample and the liquid due to too high a power, and on the other hand to ensure the penetration of the reagent into the tissue interior.

[0077] After microwave treatment, the entire experimental process can be shortened to be completed within 2 class hours. After treatment, the cytoskeleton structure is clear and complete, and the overall operation is simple, which can be popularized and used in undergraduate experiments. At the same time, the requirements for exploratory experiments in undergraduate experimental teaching are getting higher and higher, but usually exploratory experiments take a long time and the experimental design is complex. However, this experiment has a simple operation, which allows students to independently explore the optimal conditions from multiple aspects such as extraction time, fixation time, and treatment temperature, so as to cultivate students' independent experimental ability.

[0078] The above embodiments have described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A rapid preparation method for cytoskeleton experimental samples, characterized in that The specific steps are as follows: Step S1: Tear off the inner epidermis of the onion bulb and place it in a cube plastic box. Add 0.2 mol / L phosphate buffer solution. After the inner epidermis of the onion bulb sinks, place it in a microwave oven and heat it at 30 - 40 °C for 2 min. Step S2: Suck out the phosphate buffer solution, add 1 wt% Triton X-100 solution as the extraction agent to cover the onion sample, and place it in a microwave oven and heat it at 30 - 40 °C for 5 min. Step S3: Suck out the extraction agent, soak and wash it 3 times with M-buffer solution, and each time place it in a microwave oven and heat it at 30 - 40 °C for 2 min. The M-buffer solution is composed of 50 mmol / L imidazole, 50 mmol / L potassium chloride, 0.5 mmol / L magnesium chloride, 1 mmol / L EGTA, 0.1 mmol / L ethylenediaminetetraacetic acid, and 1 mmol / L β-mercaptoethanol. Step S4: Suck out the M-buffer solution, add 3 wt% glutaraldehyde solution as the fixative to cover the onion sample, and place it in a microwave oven and heat it at 30 - 40 °C for 5 min. Step S5: Suck out the fixative, soak and wash it 3 times with phosphate buffer solution, and each time place it in a microwave oven and heat it at 30 - 40 °C for 2 min. Step S6: Suck out the phosphate buffer solution, add 0.2 wt% Coomassie Brilliant Blue R250 staining solution and place it in a microwave oven and heat it at 30 - 40 °C for 3 min. Step S7: Rinse it 1 - 2 times with distilled water to remove the floating liquid, cover it with a cover glass and observe.

2. The rapid preparation method of the cytoskeleton experiment sample according to claim 1, characterized in that: The 1 wt% Triton X-100 solution as the extraction agent described in Step S2 is prepared with M-buffer solution.

3. The rapid preparation method of the cytoskeleton experimental sample according to claim 1, characterized in that: The 3 wt% glutaraldehyde solution as the fixative described in Step S4 is prepared with phosphate buffer solution.

4. The rapid preparation method of the cytoskeleton experiment sample according to claim 1, characterized in that The solvent of the 0.2 wt% Coomassie Brilliant Blue R250 staining solution described in Step S6 is composed of the following solutions by volume percentage: 46.5% methanol, 7% glacial acetic acid, and 46.5% distilled water.

5. The rapid preparation method of the cytoskeleton experimental sample according to claim 1, wherein: The sample in each step needs to be placed in a beaker filled with water when being processed in the microwave oven, and the water in the beaker should be replaced in time after each processing to avoid the temperature of the water in the beaker exceeding 40 °C.

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