A method for redyeing plant tissue sections
Through the plant tissue section re-staining method, the lignin autofluorescence is first observed, and then fluorescence and bright field staining are performed in sequence, which solves the problem of multiple staining errors of the same tissue and achieves efficient and accurate multiple staining effects.
Patent Information
- Application Number
- CN202211558778.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-06
- Publication Date
- 2025-09-05
- Estimated Expiration
- 2042-12-06
AI Technical Summary
In the existing technology, multiple staining methods for plant tissue sections need to be performed on different samples, which makes it difficult to accurately judge the differences in developmental stages, resulting in large errors in the staining results, and multiple stainings of the same tissue are prone to interference.
The plant tissue section counterstaining method was used to observe lignin autofluorescence first, and then stain with cellulose fluorescent dye and bright field dye in sequence. By using washable and non-washable dyes in a reasonable order, the interference of the previous staining step on the subsequent observation was avoided.
It enables multiple staining observations of the same sample, which is simple and quick, avoids errors caused by sample differences, and improves the accuracy and efficiency of staining results.
Smart Images
Figure CN115728118B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a plant tissue section redyeing method. Background Art
[0002] Section staining is a key technique widely used in molecular biochemical research. It utilizes dyes that interact with chemical components or structures within cells, producing distinct colors. Cell staining allows for intuitive visualization of comprehensive cellular tissue structure and analysis of the location and content of target metabolites.
[0003] Traditional cell staining methods typically use different dyes for the same target substance or cell structure, typically performed on different sample sections. This requires preparing numerous samples when multiple staining procedures are required. Furthermore, even the same plant tissue can have varying stages of development, and the boundaries between these stages are often difficult to discern. Even sections located close together can have varying stages of development and metabolic activity, easily leading to inaccuracies in staining results.
[0004] Therefore, how to provide a fast, efficient and accurate method for staining the same plant cell sections is an urgent problem to be solved in this field. Summary of the Invention
[0005] In view of this, the present invention provides a method for redyeing plant tissue sections
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] A plant tissue section redyeing method comprises the following steps:
[0008] (1) Prepare paraffin sections of plant tissue and dewax and rehydrate the paraffin sections;
[0009] (2) Observation of lignin autofluorescence on dewaxed and rehydrated plant tissue sections;
[0010] (3) staining the plant tissue sections after the observation in step (2) with a fluorescent dye for cellulose observation;
[0011] (4) The plant tissue sections after the observation in step (3) are subjected to bright field dye staining observation.
[0012] Furthermore, the dewaxing and rehydration treatment in step (1) includes: taking paraffin sections that have been placed at 37°C overnight, placing them on a slide baker at 80°C for heating and dewaxing for 12 hours, until the paraffin on the sections is basically removed; quickly putting the sections into the prepared xylene, letting them stand for 20 minutes, replacing them with new xylene, and letting them stand for 10 minutes; taking the sections out of the xylene, letting them stand vertically on absorbent paper for 10 seconds, removing the residual xylene on the glass slide, and then putting the sections into the prepared anhydrous ethanol, letting them stand for 5 minutes, repeating twice, and then putting the sections into 95% ethanol, 75% ethanol, 50% ethanol, 30% ethanol and pure water in turn, and letting each gradient stand for 3 minutes.
[0013] Furthermore, the observation in step (2) includes: using 50% glycerol to press the slices to make plant tissue slides, directly using blue light as the excitation light source under a fluorescence microscope to observe the fluorescence signal, and rinsing with distilled water after the observation is completed.
[0014] Furthermore, step (3) fluorescent dye staining observation includes: washable fluorescent dye staining observation and non-washable fluorescent dye staining observation; when both staining methods are used, the order is to first perform washable fluorescent dye staining observation and then perform non-washable fluorescent dye staining observation.
[0015] Furthermore, step (4) bright field dye staining observation includes: washable bright field dye staining observation and non-washable bright field dye staining observation; when both staining methods are used, the order is to first perform washable bright field dye staining observation and then perform non-washable bright field dye staining observation.
[0016] Furthermore, the washable fluorescent dye includes a fluorescent whitening agent;
[0017] The non-washable fluorescent dye includes Congo red;
[0018] The washable bright field dyes include phloroglucinol, safranin, and zinc iodide;
[0019] The non-washable brightfield dye includes toluidine blue.
[0020] Furthermore, the washable fluorescent dye staining observation in step (3) includes: diluting the fluorescent whitening agent stock solution by 20-50 times, adding 80 μl of the fluorescent whitening agent dilution solution and 2 drops of 10% KOH solution to the slide washed in step (2), and staining for 3-5 minutes; rinsing the slide with distilled water, pressing the slide with 50% glycerol solution, observing under ultraviolet light, and rinsing with distilled water after the observation is completed;
[0021] The non-washable fluorescent dye staining observation includes: dropping 80 μL of 0.5% Congo red staining solution on the washed slide and staining for 3-5 minutes; after gently rinsing the slide with distilled water, pressing the slide with 50% glycerol solution and observing the red fluorescence emitted by the combination of Congo red and cellulose under blue fluorescence; after the observation is completed, rinsing with distilled water, and immersing the slide in a decolorizing solution mixed with anhydrous ethanol and glacial acetic acid in a ratio of 3:1 for 30 minutes to decolorize until the section is completely colorless.
[0022] Furthermore, the washable bright field dye staining observation in step (4) includes: using 3% phloroglucinol alcohol solution and concentrated hydrochloric acid in a ratio of 2:1 to add 80 μL of phloroglucinol hydrochloric acid solution to the decolorized slide, and immediately pressing the slide; after staining for 1-3 minutes, directly observing the purple-red signal displayed by the phloroglucinol hydrochloric acid solution binding to G-type lignin through a bright field microscope, rinsing with distilled water after the observation is completed, and immersing the slide in a decolorizing solution mixed with anhydrous ethanol and glacial acetic acid in a ratio of 3:1 for 5 minutes to decolorize until the slide is completely colorless;
[0023] The non-washable brightfield dye staining observation includes: using distilled water to dilute 1% toluidine blue to a working concentration of 0.5%, aspirating 80 μL and dropping it on the decolorized slide, staining for 1-3 minutes, and stopping the staining when the band turns purple-black visibly to the naked eye; rinsing the slide with distilled water two to three times until no obvious excess dye solution remains, adding distilled water and pressing on a cover slip for observation in the bright field.
[0024] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are:
[0025] The present invention rationally prioritizes dyes based on their inherent properties and washability, prioritizing fluorescent dyes before brightfield dyes and washable dyes before non-washable dyes. This prevents the previous dyeing result from significantly interfering with subsequent dyeing observations, achieving excellent results for multiple staining observations of the same sample. Multiple staining observations of the same sample are simple and quick, avoiding errors caused by sample differences and enabling multiple observations when samples are scarce. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.
[0027] Figure 1This is the result of lignin autofluorescence staining in Example 1 of the present invention;
[0028] Figure 2 This is the dyeing result of cellulose fluorescent whitening agent in Example 1 of the present invention;
[0029] Figure 3 This is the toluidine blue staining result of Example 1 of the present invention;
[0030] Figure 4 Lignin autofluorescence staining results of Example 2 of the present invention;
[0031] Figure 5 This is the dyeing result of cellulose fluorescent whitening agent in Example 2 of the present invention;
[0032] Figure 6 This is the Congo red staining result of cellulose in Example 2 of the present invention;
[0033] Figure 7 This is the phloroglucinol staining result of Example 2 of the present invention;
[0034] Figure 8 This is the toluidine blue staining result of Example 2 of the present invention;
[0035] Figure 9 This is the toluidine blue staining result of Comparative Example 1 of the present invention;
[0036] Figure 10 This is the result of toluidine blue staining + safranin staining in comparative example 1 of the present invention;
[0037] Figure 11 This is the safranin dyeing result of Comparative Example 2 of the present invention; DETAILED DESCRIPTION
[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0039] Example 1
[0040] (1) Dewaxing and rehydrating paraffin sections: Take paraffin sections that have been placed at 37°C overnight and place them on a slide oven at 80°C for 12 hours for high-temperature dewaxing until the paraffin on the sections is basically removed. Quickly place the sections in the prepared xylene and let it stand for 20 minutes. Replace with new xylene and let it stand for 10 minutes. Take the sections out of the xylene and let it stand vertically on absorbent paper for 10 seconds. After removing the residual xylene on the glass slide, place the sections in the prepared anhydrous ethanol and let it stand for 5 minutes. Repeat twice, then place the sections in 95% ethanol, 75% ethanol, 50% ethanol, 30% ethanol and pure water in turn, and let each gradient stand for 3 minutes.
[0041] (2) Observation of lignin autofluorescence: Use 50% glycerol to press the pellet and observe the fluorescence signal directly under a fluorescence microscope using blue light as the excitation light source. The results are as follows: Figure 1 After observation, rinse with distilled water.
[0042] (3) Observation of cellulose fluorescent whitening agent staining: Dilute the commercial fluorescent whitening agent stock solution 20 times (concentration is 5%), add 80 μL of dye solution and 2 drops of 10% KOH solution to the slide cleaned in step (2) and stain for 3 minutes. After rinsing with distilled water, press the slide with 50% glycerol and observe the blue fluorescence emitted by the combination of fluorescent whitening agent and cellulose under ultraviolet light. The results are as follows: Figure 2 After observation, rinse with distilled water.
[0043] (4) Toluidine blue staining observation: dilute 1% toluidine blue with distilled water to a working concentration of 0.5%, draw 80 μL and drop it on the slide after cleaning in step (3), stain for 1 minute, and stop staining when the band turns purple-black. Rinse the slide with distilled water two to three times until there is no obvious excess dye residue, add distilled water and press the cover glass on it and observe in the bright field. The results are as follows: Figure 3 shown.
[0044] Example 2
[0045] (1) Dewaxing and rehydrating paraffin sections: Take paraffin sections that have been placed at 37°C overnight and place them on a slide oven at 80°C for 12 hours for high-temperature dewaxing until the paraffin on the sections is basically removed. Quickly place the sections in the prepared xylene and let it stand for 20 minutes. Replace with new xylene and let it stand for 10 minutes. Take the sections out of the xylene and let it stand vertically on absorbent paper for 10 seconds. After removing the residual xylene on the glass slide, place the sections in the prepared anhydrous ethanol and let it stand for 5 minutes. Repeat twice, then place the sections in 95% ethanol, 75% ethanol, 50% ethanol, 30% ethanol and pure water in turn, and let each gradient stand for 3 minutes.
[0046] (2) Observation of lignin autofluorescence: Use 50% glycerol to press the pellet and observe the fluorescence signal directly under a fluorescence microscope using blue light as the excitation light source. The results are as follows: Figure 4 After observation, rinse with distilled water.
[0047] (3) Observation of cellulose fluorescent whitening agent staining: Dilute the commercial fluorescent whitening agent stock solution 20 times (concentration is 5%), add an appropriate amount of 80 μL of dye solution and 2 drops of 10% KOH solution to the slide cleaned in step (2) and stain for 5 minutes. After rinsing with distilled water, press the slide with 50% glycerol and observe the blue fluorescence emitted by the combination of fluorescent whitening agent and cellulose under ultraviolet light. The results are as follows: Figure 5 After observation, rinse with distilled water.
[0048] Cellulose Congo red staining observation: 80 μL of 0.5% Congo red staining solution was added to the cleaned slide and stained for 5 minutes. After gently rinsing the slide with distilled water, the slide was pressed with 50% glycerol solution and the red fluorescence emitted by the combination of Congo red and cellulose was observed under blue fluorescence. The results are as follows Figure 6 As shown, after the observation, the slides were rinsed with distilled water and placed in a decolorizing solution of a mixture of anhydrous ethanol and glacial acetic acid in a ratio of 3:1 for 30 minutes until the sections were completely colorless in the bright field.
[0049] (4) Phloroglucinol staining: 80 μL of 3% phloroglucinol alcohol solution and concentrated hydrochloric acid in a ratio of 2:1 was added to the decolorized slide and immediately pressed. After staining for 1 minute, the purple-red signal displayed by the combination of phloroglucinol hydrochloric acid solution and G-type lignin was directly observed under a bright field microscope. The results are as follows: Figure 7 As shown, after the observation, rinse with distilled water and place the slide in a decolorizing solution mixed with anhydrous ethanol and glacial acetic acid in a ratio of 3:1 for 5 minutes until the slide is completely colorless.
[0050] Toluidine blue staining: dilute 1% toluidine blue with distilled water to a working concentration of 0.5%, draw 80 μL and drop it onto the decolorized slide, stain for 1 minute, and stop staining when the band turns purple-black to the naked eye; rinse the slide two to three times with distilled water until no excess dye remains, add distilled water, press on the cover glass and observe in the bright field. The results are as follows Figure 8 shown.
[0051] Comparative Example 1
[0052] (1) Dewaxing and rehydrating paraffin sections: Take paraffin sections that have been placed at 37°C overnight and place them on a slide oven at 80°C for 12 hours for high-temperature dewaxing until the paraffin on the sections is basically removed. Quickly place the sections in the prepared xylene and let it stand for 20 minutes. Replace with new xylene and let it stand for 10 minutes. Take the sections out of the xylene and let it stand vertically on absorbent paper for 10 seconds. After removing the residual xylene on the glass slide, place the sections in the prepared anhydrous ethanol and let it stand for 5 minutes. Repeat twice, then place the sections in 95% ethanol, 75% ethanol, 50% ethanol, 30% ethanol and pure water in turn, and let each gradient stand for 3 minutes.
[0053] (2) Toluidine blue staining: dilute 1% toluidine blue with distilled water to a working concentration of 0.5%, draw 80 μL and drop it onto the glass slide prepared in step (1), stain for 1 minute, and stop staining when the band turns purple-black to the naked eye; rinse the glass slide with distilled water two to three times until no obvious excess dye remains, add distilled water and press the cover glass on it and observe in the bright field. Figure 9 shown.
[0054] (3) Safranin staining: Decolorize the slides in a decolorizing solution of anhydrous ethanol and glacial acetic acid in a ratio of 3:1 for 30 minutes, then stain the slides in a 1% safranin aqueous solution for 5 minutes, rinse with distilled water, add distilled water, press on a cover glass, and observe in the bright field. The results are as follows: Figure 10 shown.
[0055] Comparative Example 2
[0056] (1) Dewaxing and rehydrating paraffin sections: Take paraffin sections that have been placed at 37°C overnight and place them on a slide oven at 80°C for 12 hours for high-temperature dewaxing until the paraffin on the sections is basically removed. Quickly place the sections in the prepared xylene and let it stand for 20 minutes. Replace with new xylene and let it stand for 10 minutes. Take the sections out of the xylene and let it stand vertically on absorbent paper for 10 seconds. After removing the residual xylene on the glass slide, place the sections in the prepared anhydrous ethanol and let it stand for 5 minutes. Repeat twice, then place the sections in 95% ethanol, 75% ethanol, 50% ethanol, 30% ethanol and pure water in turn, and let each gradient stand for 3 minutes.
[0057] (2) Safranin staining: Place the slide in a 1% safranin aqueous solution for 5 minutes, rinse with distilled water, add distilled water, press on the cover glass and observe in the bright field. The results are as follows: Figure 11 shown.
[0058] Comparative Example 2 Figure 7-8 , Comparative Example 1 Figure 10 and Comparative Example 2 Figure 11 Can discover:
[0059] Example 2 Figure 7 Comparative Example 2 Figure 11 The results of the staining are relatively close, because safranin staining only stains lignified cells, and the results should be similar to those of phloroglucinol staining. This also proves that the fluorescent dye staining step in step (3) of Example 2 does not interfere with the subsequent phloroglucinol staining. That is, in the multiple staining steps designed by this application for a single plant tissue section, placing the fluorescent dye staining before the bright field dye staining has no effect on the staining results.
[0060] Comparative Example 2 Figure 11 Comparison, Comparative Example 1 Figure 10 The stained area is abnormal, showing non-specific coloration and a purple tint in the red, which is related to the incomplete removal of the toluidine blue stain. The abnormal results of the safranin staining are caused by the incomplete decolorization of the toluidine blue stain. This also proves that placing the non-washable brightfield dye staining before the washable brightfield dye staining will cause abnormal staining results. That is, in the multiple staining steps designed for a single plant tissue section by this application, placing the washable brightfield dye staining before the non-washable brightfield dye staining has no effect on the staining results.
[0061] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for redyeing plant tissue sections, characterized in that: The following steps are involved: (1) Prepare paraffin sections of plant tissue and dewax and rehydrate the paraffin sections; (2) Observation of lignin autofluorescence on dewaxed and rehydrated plant tissue sections; (3) staining the plant tissue sections after the observation in step (2) with a fluorescent dye for cellulose observation; The fluorescent dye staining observation includes: washable fluorescent dye staining observation and non-washable fluorescent dye staining observation; when both staining methods are used, the order is to first perform washable fluorescent dye staining observation, and then perform non-washable fluorescent dye staining observation after washing; (4) After the plant tissue sections have been observed in step (3), they are washed and decolorized, and then subjected to bright field dye staining observation; The bright field dye staining observation includes: washable bright field dye staining observation and non-washable bright field dye staining observation; when both staining methods are used, the order is to first perform washable bright field dye staining observation, then perform non-washable bright field dye staining observation after washing and decolorization.
2. A plant tissue section redyeing method according to claim 1, characterized in that, The dewaxing and rehydration treatment in step (1) includes: taking paraffin sections that have been placed at 37°C overnight, placing them on a slide baker at 80°C for heating and dewaxing for 12 hours until the paraffin on the sections is removed; quickly placing the sections in the prepared xylene, letting them stand for 20 minutes, replacing them with new xylene, and letting them stand for 10 minutes; taking the sections out of the xylene, letting them stand vertically on absorbent paper for 10 seconds, removing the residual xylene on the glass slide, and then placing the sections in the prepared anhydrous ethanol, letting them stand for 5 minutes, repeating twice, and then placing the sections in 95% ethanol, 75% ethanol, 50% ethanol, 30% ethanol and pure water in sequence, and letting each gradient stand for 3 minutes.
3. A plant tissue section redyeing method according to claim 1, characterized in that, The observation in step (2) includes: using 50% glycerol to press the slices to make plant tissue slice slides, directly using blue light as the excitation light source under a fluorescence microscope to observe the fluorescence signal, and rinsing with distilled water after the observation is completed.
4. A plant tissue section redyeing method according to claim 1, characterized in that, The washable fluorescent dye includes a fluorescent whitening agent; The non-washable fluorescent dye includes Congo red; The washable bright field dyes include phloroglucinol, safranin, and zinc iodide; The non-washable brightfield dye includes toluidine blue.
5. A plant tissue section redyeing method according to claim 4, characterized in that, The washable fluorescent dye staining observation in step (3) comprises: diluting the fluorescent whitening agent stock solution by 20-50 times, adding 80 μl of the fluorescent whitening agent dilution solution and 2 drops of 10% KOH solution to the glass slide washed in step (2), and staining for 3-5 minutes; rinsing the glass slide with distilled water, pressing the slide with 50% glycerol solution, observing under ultraviolet light, and rinsing with distilled water after the observation is completed; The non-washable fluorescent dye staining observation includes: adding 80 μL of 0.5% Congo red staining solution to the washed slide and staining for 3-5 minutes; after gently rinsing the slide with distilled water, pressing the slide with 50% glycerol solution and observing the red fluorescence emitted by the combination of Congo red and cellulose under blue fluorescence; after the observation is completed, rinsing with distilled water, and immersing the slide in a decolorizing solution mixed with anhydrous ethanol and glacial acetic acid in a ratio of 3:1 for 30 minutes until the section is completely colorless.
6. A plant tissue section redyeing method according to claim 4, characterized in that, The washable bright field dye staining observation in step (4) comprises: using 3% phloroglucinol alcohol solution and concentrated hydrochloric acid in a ratio of 2:1 to add 80 μL of phloroglucinol hydrochloric acid solution to the decolorized slide, and immediately pressing the slide; after staining for 1-3 minutes, directly observing the purple-red signal displayed by the combination of the phloroglucinol hydrochloric acid solution and G-type lignin through a bright field microscope, rinsing with distilled water after the observation is completed, and immersing the slide in a decolorizing solution mixed with anhydrous ethanol and glacial acetic acid in a ratio of 3:1 for 5 minutes to decolorize until the slide is completely colorless; The non-washable brightfield dye staining observation includes: diluting 1% toluidine blue with distilled water to a working concentration of 0.5%, aspirating 80 μL and dropping it on the decolorized slide, staining for 1-3 minutes, and stopping the staining when the band turns purple-black visibly to the naked eye; rinsing the slide with distilled water two to three times until no obvious excess dye remains, adding distilled water and pressing on a cover slip for observation in the bright field.