Paraffin section pretreatment method suitable for plant spatial transcriptomics
The paraffin section pretreatment method solves the problems of permeability and signal interference in fluorescence in situ hybridization in plant tissues, enabling high-throughput gene detection. It is applicable to a variety of plant tissues, especially maize and rice.
Patent Information
- Application Number
- CN202610438646.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-03
- Publication Date
- 2026-07-10
AI Technical Summary
Existing animal fluorescence in situ hybridization technology cannot be directly applied to plant systems, mainly because the structure of plant cell walls affects probe permeability, endogenous fluorescent substances interfere with signal acquisition, RNA degradation and tissue morphology preservation are difficult, making high-throughput gene detection difficult to achieve.
The paraffin section pretreatment method, which includes sample fixation, dehydration, clearing, paraffin embedding, and section preparation, combined with cellulase and dilute hydrochloric acid treatment, enhances cell permeability, removes fluorescent interfering substances, and maintains tissue morphology, is applicable to a variety of plant tissues.
It achieves high-throughput RNA fluorescence in situ hybridization in plant tissues, enabling simultaneous detection of dozens of genes. It is suitable for single-cell and spatial transcriptome cell population identification, improves detection sensitivity, and is applicable to various plant tissues such as maize and rice.
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Figure CN122357692A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of paraffin sectioning technology, and more particularly to a paraffin section pretreatment method suitable for plant spatial transcriptomics. Background Technology
[0002] RNA in situ hybridization (ISH) is commonly used to detect spatial information about gene expression in plant organs / tissues. However, traditional ISH can only detect one gene at a time, and verifying the expression patterns of several genes via ISH is very time-consuming, especially when applied to single-cell transcriptome cell population identification. Fluorescence in situ hybridization (FISH) is a technique for visualizing transcripts in fixed cells and tissues. Probe-based RNA FISH can highly specifically target specific mRNAs and provide spatial information about gene expression at the cellular and subcellular levels.
[0003] In recent years, researchers have developed various methods to verify the spatial localization of genes in plants. Single-molecule fluorescence in situ hybridization (smFISH) is an efficient method for visualizing and quantifying single RNA molecules within intact cells. This technology can probe gene expression at the single-cell and single-molecule level, providing valuable insights for the study of cellular processes and intercellular heterogeneity. With technological improvements and developments, multiple error robust fluorescence in situ hybridization (MERFISH) has overcome the limitation of traditional smFISH, which can only detect 3-5 genes at a time, and can simultaneously detect up to 1000 genes. However, due to its high cost, there are few reports on MERFISH in plants. Half-mount and whole-mount HCR-FISH, whole-mount smFISH, and PHYTOMap are probe-based, highly sensitive mRNA in situ detection methods. However, simultaneously detecting dozens or even hundreds of transcripts using these methods remains very challenging.
[0004] The key limitations of existing fluorescence in situ hybridization techniques applicable to animals that cannot be directly applied to plant systems are as follows:
[0005] 1) Although OCT embedding, which is widely used in animal FISH, can effectively avoid RNA degradation, its application in plants is limited by factors such as plant cell wall structure (e.g., lignification, cellulose density) and the susceptibility of frozen sections to ice crystal damage.
[0006] 2) The unique cell wall structure of plants hinders probe permeation, and the protease digestion conditions of animal cells are incompatible with plant tissues;
[0007] 3) Endogenous fluorescent substances in plant tissues (such as chlorophyll / phenolic compounds) interfere with fluorescence signal acquisition;
[0008] 4) Unlike traditional ISH methods for detecting single genes, FISH experiments require multiple signal stripping-hybridization-imaging cycles of the slides to meet the needs of medium- and high-throughput in situ gene detection. As Wu et al. (Nature Biomedical Engineering, 2024, 8:872-889) proposed in their subcellular resolution spatial multi-omics technology study, the above operations have extremely high requirements for maintaining the cell morphology and mRNA stability of the tissue. Summary of the Invention
[0009] In view of this, the purpose of this invention is to provide a paraffin section pretreatment method suitable for plant spatial transcriptomics, which can be widely applied to various plant tissues and contributes to the popularization and development of high-throughput spatial transcriptomics in plants.
[0010] The present invention solves the above-mentioned technical problems through the following technical means:
[0011] This invention discloses a paraffin section pretreatment method suitable for plant spatial transcriptomics, comprising the following steps:
[0012] (1) Sample collection and fixation: Dissected samples are placed in pre-cooled PFA fixative. Depending on the sample tissue, negative pressure is applied to create a vacuum until the sample sinks below the liquid surface. Then, fresh PFA fixative is added and the sample is left overnight at 4°C.
[0013] Step (1) can accelerate the penetration of the fixative, ensure that the cell morphology and RNA are fully fixed, and reduce the breakage of chloroplast structure.
[0014] (2) Sample dehydration: Dehydrated with pre-cooled 70% ethanol, and then dehydrated with 80%, 90% and 100% ethanol at room temperature;
[0015] (3) Transparency: The sample was thoroughly transparent by passing it through solutions I and II and twice through solution III under room temperature and negative pressure conditions.
[0016] Step (3) can remove lipid-soluble fluorescent substances and effectively remove interference from fluorescence signal acquisition.
[0017] (4) Paraffin embedding: Under negative pressure, the Paraffin Wax preheated to 58℃-60℃ was replaced three times, and the wax was soaked for 2 hours each time. Finally, the plant sample was immersed in liquid paraffin and stored at 4℃ after the paraffin solidified.
[0018] Step (4) can reduce tissue damage and gene expression shift caused by subsequent section fragmentation. The 2-hour paraffin infiltration ensures that the paraffin fully penetrates the intercellular spaces.
[0019] (5) Paraffin section preparation: Cut the paraffin-embedded sample block into tissue sections, carefully place the cut tissue sections into DEPC water at 42°C for development, select suitable sections, flatten them on glass slides and bake them at 42°C overnight to obtain paraffin sections;
[0020] (6) Pretreatment of sections before RNA FISH: Immerse the paraffin sections obtained in step (5) in Histo-clear for 10 min each time, twice in total, and shake the solution to fully dewax during the process; completely rehydrate the sections by passing them through a gradient of ethanol from high to low, soften the cell walls with cellulase and dilute hydrochloric acid, and then treat them with Proteinase K to degrade the cell walls and intracellular proteins to enhance cell permeability. Then fix the morphology and RNA of the treated cells with paraformaldehyde. Finally, after dehydration, the samples can be used for subsequent RNA FISH experiments.
[0021] Preferably, in step (1), the concentration of the fixative is 4% and the negative pressure is 300-500 mmHg.
[0022] Preferably, in step (3), solution I is 50% EtOH and 50% Histo-clear, solution II is 25% EtOH and 75% Histo-clear, and solution III is 100% Histo-clear.
[0023] Preferably, the glass slide in step (5) is an adhesive glass slide or a glass slide treated with polylysine.
[0024] Preferably, the thickness of the tissue slice cut in step (5) is 7-10 μm.
[0025] Preferably, the gradient ethanol in step (6) is an ethanol solution with concentrations of 85%, 70%, 50% and 30%.
[0026] The beneficial effects of this invention are:
[0027] 1. High applicability to plant tissues: This invention fully utilizes the superior tissue morphology preservation ability and adaptability of paraffin embedding to hard tissues, and successfully applies RNA fluorescence in situ hybridization based on paraffin sections to maize female ears, root tips, grains, and rice inflorescence meristems.
[0028] 2. Facilitates subsequent medium- and high-throughput in situ gene detection: Treatment with cellulase and dilute hydrochloric acid-assisted proteinase K enhances cell permeability, allowing multi-gene probes to enter cells while maintaining stable cell morphology, thus enabling the detection of dozens of genes simultaneously. This will broaden the application of plant RNA fluorescence in situ hybridization, for example, in single-cell and spatial transcriptome cell population identification.
[0029] 3. This invention has been successfully applied to RNA FISH in various plant tissues, and the detection sensitivity can reach the visualization of low-expression genes (FPKM≤20), which helps to popularize and develop plant spatial transcriptomics. Attached Figure Description
[0030] Figure 1 This is a diagram showing the results of in-situ detection of KNOTTED1 (KN1) in the meristem of maize female inflorescence using FISH processed according to the present invention.
[0031] Figure 2 This is a diagram showing the results of in situ detection of the low-expression gene BRANCHEDSILKLESS1 (BD1) in maize female inflorescence meristem using FISH processed according to the present invention.
[0032] Figure 3 This is a graph showing the results of in situ detection of known genes in multiple tissues using FISH processed according to the present invention. Detailed Implementation
[0033] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0034] Example 1
[0035] In situ detection of KNOTTED1 (KN1) in maize female inflorescence meristems was performed using the paraffin section pretreatment method described above, including the following steps:
[0036] (1) Sample collection and fixation: The dissected corn female ears were placed in pre-cooled 4% PFA fixative, and a negative pressure of 500 mmHg was applied to vacuum the corn female ears until they sank to the surface of the liquid. The fixative was then replaced and left overnight at 4°C.
[0037] (2) Sample dehydration: Dehydrated with pre-cooled 70% ethanol; dehydrated at room temperature with 80%, 90%, and 100% ethanol;
[0038] (3) Transparency: Under room temperature and negative pressure conditions, the female ears of corn are thoroughly transparent by sequentially passing through 50% EtOH / 50% Histo-clear, 25% EtOH / 75% Histo-clear, 100% Histo-clear, and 100% Histo-clear.
[0039] (4) Paraffin embedding: Under negative pressure, the paraffin wax was replaced three times, preheated to 58℃-60℃, and each time it was immersed for 2 hours. Finally, the corn ears were immersed in liquid paraffin and stored at 4℃ after the paraffin solidified.
[0040] (5) Paraffin section preparation: The paraffin-embedded maize female ears were cut into 10 μm thick sections using a microtome. The cut tissue sections were carefully placed in DEPC water at 42°C for expansion. Suitable female ear sections were selected, flattened on an adhesive glass slide, and baked overnight in a 42°C slide dryer.
[0041] (6) Pretreatment of sections before RNA FISH: Immerse the paraffin sections obtained in step (5) in Histo-clear for 10 min each time, twice in total, and shake the solution to fully dewax during the process; completely rehydrate by passing through a gradient of ethanol from high to low, then treat with 15 mg / ml cellulase and 0.2 M HCl, then with 1 ug / ml Proteinase K, then fix the cell morphology and RNA with paraformaldehyde, and finally dehydrate by passing through a gradient of ethanol. The samples are then ready for subsequent RNA FISH experiments.
[0042] (7) RNA FISH experiment: The overnight hybridization, ligase reaction, rolling circle amplification and fluorescent probe hybridization reaction were performed sequentially according to the method described by Wu et al. (Nature Biomedical Engineering, 2024, 8:872-889). The cell nuclei were stained with DAPI and then imaged under a laser confocal microscope.
[0043] Example 2
[0044] In situ detection of BRANCHED SILKLESS1 (BD1) in maize female inflorescence meristems was performed using the paraffin section pretreatment method described above, including the following steps:
[0045] (1) Sample collection and fixation: Dissected maize female ears were placed in pre-cooled 4% PFA fixative, and a negative pressure of 500 mmHg was applied to vacuum the sample until it sank to the surface of the liquid. The fixative was then replaced and left overnight at 4°C.
[0046] (2) Sample dehydration: Dehydrated with pre-cooled 70% ethanol; dehydrated at room temperature with 80%, 90%, and 100% ethanol;
[0047] (3) Transparency: Under room temperature and negative pressure conditions, the female ears of corn are thoroughly transparent by sequentially passing through 50% EtOH / 50% Histo-clear, 25% EtOH / 75% Histo-clear, 100% Histo-clear, and 100% Histo-clear.
[0048] (4) Paraffin embedding: Under negative pressure, the paraffin wax was replaced three times, preheated to 58℃-60℃, and each time it was immersed for 2 hours. Finally, the corn ears were immersed in liquid paraffin and stored at 4℃ after the paraffin solidified.
[0049] (5) Paraffin section preparation: The paraffin-embedded maize female ears were cut into 10 μm thick sections using a microtome. The cut tissue sections were carefully placed in DEPC water at 42°C for expansion. Suitable female ear sections were selected, flattened on an adhesive glass slide, and baked overnight in a 42°C slide dryer.
[0050] (6) Pretreatment of sections before RNA FISH: Immerse the paraffin sections obtained in step (5) in Histo-clear for 10 min each time, twice in total, and shake the solution to fully dewax during the process; completely rehydrate by passing through a gradient of ethanol from high to low, treat with 15 mg / ml cellulase and 0.2 M HCl, then treat with 1 ug / ml Proteinase K, fix the cell morphology and RNA with paraformaldehyde, and finally dehydrate by passing through a gradient of ethanol. The samples are then ready for subsequent RNA FISH experiments.
[0051] (7) RNA FISH experiment: The overnight hybridization, ligase reaction, rolling circle amplification, and fluorescent probe hybridization reaction were performed sequentially according to the method described in (Wu et al., 2024). The cell nuclei were stained with DAPI and then imaged under a laser confocal microscope.
[0052] Example 3
[0053] The above-described paraffin section pretreatment method for in situ detection of known genes in multiple tissues includes the following steps:
[0054] (1) Sample collection and fixation: Dissected maize root tips, kernels and rice inflorescence meristems were placed in pre-cooled 4% PFA fixative, and a negative pressure of 400 mmHg was applied to vacuum the sample until it sank to the surface of the liquid. The fixative was then replaced and left overnight at 4°C.
[0055] (2) Sample dehydration: Dehydrated with pre-cooled 70% ethanol; dehydrated at room temperature with 80%, 90%, and 100% ethanol;
[0056] (3) Transparency: The sample was thoroughly transparent by passing it through 50% EtOH / 50% Histo-clear, 25% EtOH / 75% Histo-clear, 100% Histo-clear, and 100% Histo-clear solutions sequentially under negative pressure at room temperature.
[0057] (4) Paraffin embedding: Under negative pressure, the paraffin wax was replaced three times, preheated to 58℃-60℃, and the wax was soaked for 2 hours each time. Finally, the sample was immersed in liquid paraffin and stored at 4℃ after the paraffin solidified.
[0058] (5) Paraffin section preparation: The paraffin-embedded sample was cut into 10 μm thick sections using a microtome. The cut tissue sections were carefully placed in DEPC water at 42°C for development. Suitable sections were selected, flattened on an adhesive glass slide, and baked overnight in a 42°C slide dryer.
[0059] (6) Pretreatment of sections before RNA FISH: Immerse the paraffin sections obtained in step (5) in Histo-clear for 10 min each time, twice in total, and shake the solution to fully dewax during the process; completely rehydrate by passing through a gradient of ethanol from high to low, treat with 15 mg / ml cellulase and 0.2 M HCl, then treat with 1 ug / ml Proteinase K, fix the cell morphology and RNA with paraformaldehyde, and finally dehydrate by passing through a gradient of ethanol. The samples are then ready for subsequent RNA FISH experiments.
[0060] (7) RNA FISH experiment: according to The method involved overnight hybridization, ligase reaction, rolling circle amplification, and fluorescent probe hybridization. Cell nuclei were stained with DAPI and then imaged under a laser confocal microscope.
[0061] Figure 1 This image shows the results of in situ detection of KNOTTED1 (KN1) in maize female inflorescence meristem using FISH preprocessing according to this invention. The expression pattern of KN1 was visualized in longitudinal sections of maize female ears using FISH (A) and traditional in situ hybridization (B). The results showed that the KN1 expression pattern was comparable to that of traditional in situ hybridization, indicating high expression in the apical inflorescence meristem. Scale bar: 100 μm.
[0062] Figure 2 This image shows the results of in situ detection of the low-expression gene BRANCHED SILKLESS1 (BD1) in maize female inflorescence meristem using FISH pretreated according to this invention. The expression pattern of BD1 was visualized in longitudinal sections of maize female ears using FISH (A) and conventional in situ hybridization (B), revealing that BD1 was specifically expressed at the meristem boundary. Scale bar: 100 μm.
[0063] Figure 3This image shows the results of in situ detection of known genes in maize root tips, kernels, and rice inflorescence meristems using FISH pretreated according to this invention, demonstrating that the pretreated FISH is applicable to different plant tissues. ZmGLYCINE RICH PROTEIN4 (ZmGRP4) expression was detected in root cap cells in a maize root tip cross-section (A); AL9 specific expression in the aleurone layer was detected in kernels 6 days after pollination (B); and LAX PANICLE1 (LAX1) expression was detected in the meristem boundary region in rice inflorescence meristems (C). Scale bar: 100 μm.
[0064] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.
Claims
1. A paraffin section pretreatment method suitable for plant spatial transcriptomics, characterized in that, Includes the following steps: (1) Sample collection and fixation: Dissected samples are placed in pre-cooled PFA fixative. Depending on the sample tissue, negative pressure is applied to create a vacuum until the sample sinks below the liquid surface. Then, fresh PFA fixative is used and the sample is left overnight at 4°C. (2) Sample dehydration: Dehydrated with pre-cooled 70% ethanol, and then dehydrated with 80%, 90% and 100% ethanol at room temperature; (3) Transparency: The sample was thoroughly transparent by passing it through solutions I and II and twice through solution III under room temperature and negative pressure conditions. (4) Paraffin embedding: Under negative pressure, the Paraffin Wax preheated to 58℃-60℃ was replaced three times, and the wax was soaked for 2 hours each time. Finally, the plant sample was immersed in liquid paraffin in the embedding frame and stored at 4℃ after the paraffin solidified. (5) Paraffin section preparation: Cut the paraffin-embedded sample block into tissue sections, carefully place the cut tissue sections into DEPC water at 42°C for development, select suitable sections, flatten them on a glass slide, and bake them at 42°C overnight to obtain paraffin sections. (6) Pretreatment of sections before RNA FISH: Immerse the paraffin sections obtained in step (5) in Histo-clear for 10 min each time, twice in total, and shake the solution to fully dewax during the process; completely rehydrate by passing through a gradient of ethanol from high to low, then treat with 15 mg / ml cellulase and 0.2 M HCl, then treat with 1 ug / ml Proteinase K, then fix the cell morphology and RNA with paraformaldehyde, and finally dehydrate the samples before RNA FISH experiments.
2. The paraffin section pretreatment method for plant spatial transcriptomics according to claim 1, characterized in that, In step (1), the concentration of the fixative is 4% and the negative pressure is 300-500 mmHg.
3. The paraffin section pretreatment method for plant spatial transcriptomics according to claim 1, characterized in that, In step (3), solution I is 50% EtOH and 50% Histo-clear, solution II is 25% EtOH and 75% Histo-clear, and solution III is 100% Histo-clear.
4. The paraffin section pretreatment method for plant spatial transcriptomics according to claim 1, characterized in that, The glass slide in step (5) is an adhesive glass slide or a glass slide treated with polylysine.
5. The paraffin section pretreatment method for plant spatial transcriptomics according to claim 1, characterized in that, The thickness of the tissue slice cut in step (5) is 7-10 μm.
6. The paraffin section pretreatment method for plant spatial transcriptomics according to claim 1, characterized in that, The gradient ethanol in step (6) consists of ethanol concentrations of 85%, 70%, 50%, and 30%.