Method for animal vascular tissue cryo-embedding and sectioning and spatial transcriptome sequencing

Abstract

The application belongs to the technical field of frozen section, and particularly relates to an animal blood vessel tissue frozen embedding and sectioning and spatial transcriptome sequencing method. In the first aspect, the application provides an animal blood vessel tissue frozen embedding method, wherein a first buffer is used to replace blood in an animal body; a blood vessel is peeled off and surrounding tissues are removed; a perfusion liquid is used to perfuse the blood vessel to obtain a perfused blood vessel, the perfusion liquid comprising OCT and a second buffer; and a plurality of perfused blood vessels are embedded in the same OCT embedding block and then rapidly frozen. The blood vessel is peeled off from the tissue and embedded alone, and a plurality of blood vessels in each embedding block are laid flat after sectioning to ensure that the chip surface is covered with sufficient blood vessel structures, thereby solving the problems of small volume of blood vessel tissue, small number of cells, small amount of RNA and difficulty in spatial transcriptome sequencing and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of frozen section, and particularly relates to an animal blood vessel tissue frozen embedding and sectioning and spatial transcriptome sequencing method. BACKGROUND

[0002] Vessels are important organizational structures that undertake blood transport, nutrition and metabolic exchange in the human body, and are also important transport pathways for the circulation of the body. At the same time, blood vessels directly affect the health status of the human body, and abnormal blood vessels can cause many problems, such as allergic diseases, immune system diseases, metabolic syndrome, cardiovascular and cerebrovascular diseases, and aortic cancer. Especially in the aspect of cardiovascular and cerebrovascular diseases, arteriosclerosis, gout, thrombotic occlusive vasculitis, coronary heart disease, stroke and other diseases are closely related to blood vessel function. Therefore, comprehensively understanding the cell type composition, spatial structure localization and special structure and function of blood vessels can help further understand the function of living organisms and the mechanism of related diseases.

[0003] In scientific research related to blood vessels, mice are the most common model organisms, and the scientific research community widely uses mouse blood vessels to study human-related vascular diseases. At present, single-cell sequencing and the newly developed spatial transcriptome technology are commonly used. Some scholars have clustered and analyzed the expression of aortic cell genes, identified a variety of cell types, including fibroblasts, vascular smooth muscle cells, endothelial cells and immune cells (monocytes, macrophages and lymphocytes), and drawn a detailed single-cell map. However, although single-cell sequencing technology can detect the biological characteristics of individual cells and reveal the heterogeneity between cells, important spatial location information is lost because the tissue is dissociated into single cells. The emerging spatial transcriptome can detect nucleic acid molecules in tissue section cells in situ in space, further reveal the spatial structure and functional heterogeneity between tissue cells in space while identifying cell types, and pioneeringly draw the spatial single-cell transcriptome map of blood vessels, comprehensively understand the characteristics and relationship between the macro-micro structure and function of blood vessels, and further reveal the molecular mechanism of the occurrence of blood vessel-related diseases.

[0004] The most widely used technology in spatial transcriptome technology is the technology of marking the spatial tag information of nucleic acid information (in situ barcoding based methods). This technology captures the mRNA in situ labeling of fresh frozen tissue sections, and through reverse transcription, amplification, library construction, sequencing, alignment analysis and other steps, the complete tissue section panoramic spatial transcriptome map can be obtained. This technology includes spatial transcriptomis (ST), which was optimized by 10xGenomics company in 2017 and launched the commercial Visium kit, Slide-seq, Slide-seq V2, HDST, DBiT-seq, Pixel-seq, Seq-Scope, and the Stereo-seq technology independently developed by Huada. In this type of spatial transcriptome technology, the most critical pre-processing step is the freezing embedding and sectioning experiment of the blood vessel tissue. Once the high-quality, ideal and intact tissue section cannot be obtained, the subsequent spatial transcriptome experiment cannot be carried out, or even if the experiment can be carried out, the high-quality sequencing result cannot be obtained, and the further biological analysis cannot be carried out, which declares the failure of the whole experiment. Therefore, the establishment of the embedding and sectioning scheme of the blood vessel tissue is very important.

[0005] At present, the scheme of exploring the spatial transcriptome expression characteristics related to blood vessels by using spatial transcriptome technology is to process and embed the whole tissue at the same time, obtain the cross section of the whole tissue by sectioning, detect the transcriptome expression, and finally identify the blood vessel tissue by auxiliary means and carry out independent research on the blood vessel. This scheme has very low experimental efficiency, and only one cross section experiment can be carried out at a time. Most of the information obtained is from the tissue outside the blood vessel, and the effective data rate is low. At the same time, the process of obtaining the cross section needs constant adjustment of the cross section, which consumes a lot of time and energy, and the experimental cost is high. At present, there is no spatial transcriptome experiment on the blood vessels separated and embedded and sectioned. However, in the process of research and practice, it is found that this scheme has the following shortcomings which need to be optimized:

[0006] 1) The volume of blood vessel tissue is small, and after 10 microns sectioning, the number of cells is small, and the nucleic acid information obtained is less. The RNA yield of fresh frozen tissue section of a single sample is difficult to meet the requirements of subsequent spatial transcriptome experiment, and it is difficult to capture sufficient nucleic acid information for subsequent analysis;

[0007] 2) The blood vessel tissue structure is hollow, and it is easy to deform due to extrusion of external reagents during the conventional freezing embedding scheme;

[0008] 3) The separation of mouse blood vessels requires a certain time, and during this process, RNA degradation is easy to occur, which reduces the RNA quality and makes the spatial transcriptome experiment unable to capture sufficient nucleic acid information;

[0009] 4) The cell composition, tissue state, etc. of blood vessel tissue are different from those of large tissues such as brain and heart. Various conditions such as sectioning temperature and sectioning thickness need to be tested and adjusted to obtain the best sectioning conditions, and finally obtain tissue sections suitable for spatial transcriptome experiments, which are not fragmented, not wrinkled, complete in shape, single-layer cells, and high in RNA quality. SUMMARY

[0010] The first aspect of the present application aims to provide a method for cryo-embedding animal blood vessel tissue.

[0011] The second aspect of the present application aims to provide a sectioning method for animal blood vessel tissue.

[0012] The third aspect of the present application aims to provide a method for spatial transcriptome sequencing.

[0013] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0014] The first aspect of the present application provides a method for cryo-embedding animal blood vessel tissue, comprising the following steps:

[0015] The blood in the animal body is replaced with a first buffer solution;

[0016] The blood vessel is peeled off and the surrounding tissue is removed;

[0017] The blood vessel is perfused with a perfusion solution to obtain a perfused blood vessel, wherein the perfusion solution comprises OCT and a second buffer solution;

[0018] The plurality of perfused blood vessels are embedded in the same OCT embedding block and then rapidly frozen.

[0019] Preferably, the number of blood vessels embedded in the same OCT embedding block is 2-20.

[0020] Preferably, the number of blood vessels embedded in the same OCT embedding block is 2-10.

[0021] Preferably, the volume percentage of OCT in the perfusion solution is less than 70%.

[0022] Preferably, the volume percentage of OCT in the perfusion solution is 10-60%.

[0023] Preferably, the volume percentage of OCT in the perfusion solution is 40-60%.

[0024] Preferably, the volume percentage of OCT in the perfusion solution is 45-55%.

[0025] Preferably, the perfusion solution further comprises an RNAase inhibitor.

[0026] Preferably, the perfusion solution comprises OCT, a second buffer and an RNAse inhibitor.

[0027] Preferably, the volume percentage of the RNAse inhibitor in the perfusion solution is 1-10%.

[0028] Preferably, the volume percentage of the OCT in the perfusion solution is less than 70%, and the volume percentage of the RNAse inhibitor is 1-10%.

[0029] Preferably, the volume percentage of the OCT in the perfusion solution is 40-60%, and the volume percentage of the RNAse inhibitor is 4-7%.

[0030] Preferably, the animal is a rodent.

[0031] Preferably, the animal is a mouse or a rat.

[0032] Preferably, the first buffer and the second buffer are independently selected from at least one of physiological saline and PBS buffer.

[0033] Preferably, the blood vessel is selected from at least one of an artery, a vein and a microvessel.

[0034] Preferably, the artery is selected from at least one of an elastic artery, a muscular artery and a small artery.

[0035] Preferably, the vein is selected from at least one of a large vein, a medium vein and a small vein.

[0036] Preferably, the microvessel is selected from at least one of a continuous microvessel and a permeable microvessel.

[0037] Preferably, the elastic artery is selected from at least one of a thoracic aorta, an abdominal aorta, a subclavian artery and a common carotid artery.

[0038] In a second aspect, the present application provides a method for sectioning animal vascular tissue, comprising the following steps:

[0039] freezing and embedding the animal vascular tissue according to the method for freezing and embedding animal vascular tissue as described above;

[0040] sectioning the frozen and embedded tissue.

[0041] Preferably, the thickness of the section is 6-14 microns.

[0042] Preferably, the temperature of the sample head during sectioning is -15 to -10℃.

[0043] In a third aspect, the present application provides a method for spatial transcriptome sequencing, comprising the following steps:

[0044] Freezing embedding the animal blood vessel tissue according to the foregoing animal blood vessel tissue freezing embedding method;

[0045] Slicing the freezing embedded tissue;

[0046] Spatial transcriptome sequencing is performed on the slices.

[0047] Preferably, when the spatial transcriptome sequencing is performed on the slices, a plurality of the slices are sequentially tiled and combined on a spatial transcriptome sequencing chip.

[0048] Preferably, the number of the slices sequentially tiled and combined on the spatial transcriptome sequencing chip is 2-5.

[0049] Preferably, before the spatial transcriptome sequencing, a step of quality inspection of the slices is further included, and the quality inspection includes at least one of RNA integrity detection and tissue morphology detection.

[0050] The present application has the following advantages:

[0051] (1) The blood vessel tissue has a small volume, a small number of cells, and a small amount of RNA contained, and it is very difficult to perform spatial transcriptome sequencing and other related sequencing work. Therefore, in the present application, the blood vessels are stripped out of the tissue and embedded separately, a plurality of blood vessels are embedded in each embedding block, and after slicing, a plurality of slices are tiled and combined after trimming to ensure that a sufficient number of blood vessel structures are covered on the surface of a spatial transcriptome chip. In the test of the present application, a plurality of blood vessel tissue slices tiled on a spatial transcriptome chip surface can normally complete the spatial transcriptome experiment.

[0052] (2) The blood vessel tissue structure is hollow, and when a conventional freezing embedding scheme is performed, deformation is easily caused by extrusion of external reagents. Therefore, the solution adopted in the present application is to strip the blood vessels out of the tissue, perform perfusion with OCT-containing perfusion fluid, and after the blood vessels are perfused as much as possible, freezing embedding is performed, and the blood vessel structure is observed to be very full.

[0053] (3) Since the separation of mouse blood vessels requires a certain amount of time, RNA degradation easily occurs during this process, which reduces the RNA quality and makes it impossible to capture sufficient nucleic acid information in the spatial transcriptome experiment. Therefore, an RNAase inhibitor is added to the liquid contacted by the blood vessels, and the time of exposure of the blood vessels to air is strictly controlled to be no more than 2h, and RNA integrity detection is performed.

[0054] (4) It is difficult to obtain complete slices in the mouse blood vessel slicing step, and the present application optimizes the slicing temperature and slicing thickness and other parameters through a large number of tests to obtain the optimal slicing conditions for mouse blood vessel frozen sections. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1These are images of mouse aortic vessel separation from some embodiments of this application.

[0056] Figure 2 These are stained images of mouse aortic perfusion sections perfused with 30% and 50% OCT / PBS perfusion solutions in some embodiments of this application.

[0057] Figure 3 This is a stained section image of five vascular tissues embedded in an OCT embedding block in some embodiments of this application.

[0058] Figure 4 These are RIN value detection images of three slicing schemes in some embodiments of this application.

[0059] Figure 5 These are HE staining images of slices of different thicknesses from some embodiments of this application.

[0060] Figure 6 These are mouse ssDNA staining images from some embodiments of this application.

[0061] Figure 7 This is a distribution map of mouse vascular spatial transcriptome cDNA fragments in some embodiments of this application. Detailed Implementation

[0062] In the description of this invention, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first," "second," or "third" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0063] A first aspect of the present invention provides a method for cryoembedding animal vascular tissue, comprising the following steps:

[0064] The blood in the animal was replaced using the first buffer solution;

[0065] Dissect the blood vessels and remove the surrounding tissue;

[0066] The blood vessel is perfused with a perfusion solution to obtain a perfused blood vessel, wherein the perfusion solution includes OCT and a second buffer solution;

[0067] Multiple perfused blood vessels were embedded in the same OCT embedding block and then rapidly frozen.

[0068] As used herein, the term "animal" generally refers to an animal having a circulatory system comprising blood vessels, such as a vertebrate animal, including animals of the classes Agnatha, Chondrichthyes, Osteichthyes, Amphibia, Reptilia, Aves, and Mammalia. In some embodiments, the animal is a mammal (e.g., Monotremata, Marsupialia, Dasyuromorphia, Tarsiiformes, Scandentia, Dermoptera, Pteropodidea, Primates, Paenungulata, Perissodactyla, Cetacea, Artiodactyla, etc.) used for studying the physiological characteristics of blood vessels, and the pathogenesis of various blood vessel-related diseases. In some embodiments, the animal is a rodent (e.g., mouse, rat, hamster, guinea pig), a lagomorph (e.g., rabbit), an odd-toed ungulate (e.g., horse), an even-toed ungulate (e.g., sheep, pig), a primate (e.g., monkey), or a carnivore (e.g., dog).

[0069] As used herein, the term "blood vessel" generally refers to a conduit for transporting blood in an animal. Blood vessels can be generally classified as arteries, veins, and capillaries based on the direction of blood flow. Arteries transport blood from the heart to tissues throughout the body, while veins transport blood from tissues back to the heart. Capillaries connect arteries and veins and are the primary site for exchange of substances between blood and tissues. In some embodiments, the artery is selected from at least one of an elastic artery, a muscular artery, and a small artery. The elastic artery is the largest artery in diameter and is closest to the heart, and is selected from at least one of the thoracic aorta, the abdominal aorta, the subclavian artery, and the common carotid artery. The muscular artery is smaller in diameter than the elastic artery and is a branch of the elastic artery, and is selected from at least one of the femoral artery, the ulnar artery, and the radial artery. The small artery is the smallest artery in diameter, and is selected from at least one of the arteria zonae diaphysealis, the arteria afferens glomeruli, and the arteria efferens glomeruli. In some embodiments, the vein is selected from at least one of a large vein, a medium vein, and a small vein. In some embodiments, the capillary is selected from at least one of a continuous capillary and a permeable capillary.

[0070] Freezing embedding refers to the way of making the tissue quickly frozen and hardened by embedding, avoiding the problems caused by high temperature and other factors during paraffin embedding. Cells will not shrink significantly and will be close to their original shape. OCT (optimal cutting temperature compound) is a mixture of polyethylene glycol and polyvinyl alcohol, which is well known in the art. It is used to support the tissue during freezing embedding and subsequent frozen sectioning, increase the continuity of the tissue, and reduce wrinkles and fragmentation. In conventional spatial transcriptome experiments, the method of freezing embedding using OCT is to place the pretreated tissue in an embedding box containing OCT, supplement the OCT to cover the tissue, and use a freezing agent (such as liquid nitrogen, isopentane, dry ice, carbon dioxide gas, aerosol spray, etc.) to freeze at a preset temperature. After freezing, it is transferred to an environment such as -80°C for storage. Due to the particularity of vascular tissue, including its small volume, after sectioning, the number of cells and the obtained nucleic acid information are less, which is difficult to meet the requirements of subsequent spatial transcriptome experiments; at the same time, the hollow structure of the vascular tissue is easily deformed due to external agent extrusion during the conventional freezing embedding scheme; and the separation of blood vessels requires a certain time, which is easy to cause RNA degradation during this process, reducing the RNA quality and making the spatial transcriptome experiment unable to capture sufficient nucleic acid information; and the cell composition and tissue state of vascular tissue are different from those of large tissues such as brain and heart, and various conditions such as sectioning temperature and sectioning thickness still need to be tested and adjusted to obtain the best sectioning conditions. Therefore, the above method is proposed.

[0071] In some embodiments, the blood in the animal is replaced with the first perfusion fluid by flushing the blood out of the animal after dissection of the animal, typically by using the natural blood circulation system to flush the blood out of the animal through whole body perfusion via the heart or through perfusion of a nutrient artery. Specifically, the whole body perfusion via the heart includes exposing the heart, inserting a needle from the left ventricle, opening the right atrium, injecting the first buffer solution to replace the blood, and stopping until all organs are white. At this time, the blood is basically replaced by the first buffer solution and flows out from the right auricle. In some specific embodiments, the first buffer solution used for replacement includes at least one of phosphate buffered saline (PBS), normal saline, or other buffer solutions known in the art that can be used for replacement. It can be understood that, in order to avoid blood coagulation affecting replacement, the first buffer solution can also contain an appropriate amount of heparin sodium. In order to avoid affecting the operation, the first buffer solution can also contain an appropriate amount of procaine hydrochloride and other local anesthetics. Before exposing the heart, the steps of anesthetizing or euthanizing the animal are usually included, such as anesthetizing by intraperitoneal injection of sodium pentobarbital, or euthanizing by inhaling carbon dioxide.

[0072] After the replacement is completed, the blood vessel needs to be dissected, including dissection of the blood vessel, removal of its branches (if any) and surrounding tissues such as lymph nodes, so as to obtain the blood vessel as the research subject. Taking the aorta as an example, in some specific embodiments, the method for dissecting the aorta includes removing the intestine, stomach, spleen, pancreas and liver, removing the lung and flushing the chest cavity with a buffer solution, removing the abdominal aorta to drain the lymph nodes and kidneys, and then dissecting the aorta. In some of these embodiments, the aorta includes at least one of the thoracic aorta and the abdominal aorta. The thoracic aorta and the abdominal aorta can be dissected in a manner selected from the group consisting of starting from the heart and dissecting down from the esophagus and the vertebrae to the iliac bifurcation to dissect the aorta. In some of these embodiments, the surrounding structures of the aorta include arterial branches (such as the carotid artery, the subclavian artery, the celiac trunk, the mesentery and the renal artery) and lymph nodes.

[0073] Subsequently, the blood vessel is perfused with the perfusion solution formed by mixing the OCT and the second buffer solution, to obtain a perfused blood vessel, so as to expand the blood vessel to make it fuller, avoid deformation of the blood vessel due to external extrusion during subsequent embedding and slicing, and cause less damage to the original spatial structure and morphology of the blood vessel. In some specific embodiments, the volume percentage of the OCT in the perfusion solution is less than 70%, for example, it can be 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, etc. In some specific embodiments, the volume percentage of the OCT in the perfusion solution is 10-60%, 15-60%, 20-60%, 25-60%, 30-60%, 35-60%, 40-60%, 40-55%, 45-55%. In some specific embodiments, the second buffer solution includes at least one of a phosphate buffered saline (PBS), a physiological saline or other buffer solution known in the art that can be used to dilute the OCT without affecting the perfusion effect thereof. In some specific embodiments, the perfusion solution is composed of the OCT, the second buffer solution and the RNAase inhibitor, so that the content of other components can be determined accordingly.

[0074] After obtaining the perfused aortic blood vessels, the perfused blood vessels are further embedded by OCT embedding method and then frozen rapidly. In some specific embodiments, the step of embedding the perfused blood vessels by OCT includes laying OCT in the embedding box in advance, placing the perfused blood vessels in the embedding box, embedding the perfused blood vessels by OCT to form an OCT embedding block, freezing the OCT embedding block rapidly to make the OCT completely solidify, and then storing. In some embodiments, the OCT in the embedding box is laid in advance to make the OCT solidify rapidly, and then the perfused blood vessels are placed in the embedding box and embedded by OCT. In some embodiments, the freezing temperature is -25 to -15°C, for example, -25°C, -24°C, -23°C, -22°C, -21°C, -20°C, -19°C, -18°C, -17°C, -16°C, or -15°C. The storage temperature after freezing is -100 to -60°C, for example, -100°C, -99°C, -98°C, -97°C, -96°C, -95°C, -94°C, -93°C, -92°C, -91°C, -90°C, -89°C, -88°C, -87°C, -86°C, -85°C, -84°C, -83°C, -82°C, -81°C, -80°C, -79°C, -78°C, -77°C, -76°C, -75°C, -74°C, -73°C, -72°C, -71°C, -70°C, -69°C, -68°C, -67°C, -66°C, -65°C, -64°C, -63°C, -62°C, -61°C, or -60°C, usually -80°C.

[0075] Due to the small volume of the blood vessel tissue, the small number of cells, and the small amount of RNA contained, the perfused blood vessels are frozen rapidly after being embedded in the same OCT embedding block in the scheme of the present application. It can be understood that the different perfused blood vessels are independent of each other and do not interfere with each other when being embedded in the same OCT embedding block, for example, the perfused blood vessels are arranged in parallel or approximately parallel in the same OCT embedding block without crossing each other, and more tissues are attached in one chip at a time by this regular arrangement. In some specific embodiments, the number of aortic blood vessels embedded in the same OCT embedding block is 2 to 20, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the number of aortic blood vessels embedded in the same OCT embedding block is 2 to 10, 2 to 8, 3 to 8, 3 to 6, or 4 to 6.

[0076] In some embodiments, to reduce the degradation of RNA and the quality of RNA when the blood vessels are separated, and to prevent the spatial transcriptome experiment from capturing sufficient nucleic acid information and obtaining high-quality cDNA, an RNAase inhibitor is added to the perfusion solution. In some embodiments, the RNAase inhibitor includes at least one of diethyl pyrocarbonate (DEPC), guanidine isothiocyanate, vanadyl ribonucleoside complex, protein inhibitors (such as Ambion TM RNase inhibitor, RNase Inhibitor, Murine, etc.), sodium dodecyl sulfate (SDS), urea, diatomite, etc. In some embodiments, the perfusion solution contains 1-10% of the RNAase inhibitor by volume. Specifically, the RNAase inhibitor can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% by volume in the perfusion solution. It can be understood that, for laboratory environment, an RNA inhibitor such as RNase Zap can also be used, and the separation step can be accelerated and the time for which the blood vessels are exposed to air can be shortened, for example, to within 2 hours.

[0077] In a second aspect of the present application, a sectioning method of animal blood vessel tissue is provided, which includes obtaining the frozen embedded tissue according to the aforementioned frozen embedding method of animal blood vessel tissue, and then sectioning the frozen embedded tissue. In some embodiments, the frozen embedded tissue needs to be balanced before sectioning. It can be understood that, before sectioning, other instruments and equipment involved in the sectioning process, such as the sectioning machine box, brush, blade, centrifuge tube, tweezers, etc., also need to be pre-cooled to ensure temperature balance during the experiment.

[0078] Because the cell composition and tissue state of the blood vessel tissue are different from those of large tissues such as brain and heart, various parameters including sectioning temperature and sectioning thickness will affect the morphology and RNA quality of the obtained section. In some embodiments, the temperature of the sample head during sectioning is -15 to -10°C. In some embodiments, the thickness of the section is controlled to be 6-14 microns during sectioning. When the above conditions are met, the tissue section suitable for spatial transcriptome experiment can be finally obtained, which is not fragmented, not wrinkled, has complete morphology, single-layer cells, and high RNA quality. Specifically, the temperature of the sample head during sectioning can be -15°C, -14°C, -13°C, -12°C, -11°C, or -10°C. The thickness of the section is controlled to be 6 microns, 7 microns, 8 microns, 9 microns, 10 microns, 11 microns, 12 microns, 13 microns, or 14 microns during sectioning. In some embodiments, the temperature of the sample head during sectioning is -14 to -12°C. In some embodiments, the thickness of the section is controlled to be 7-13 microns, 8-12 microns, or 9-11 microns during sectioning.

[0079] In some embodiments, the thickness of the first slice is 20-60 μm, such as 50 μm. After the outline of the tissue is shown, the thickness of the slice is changed to 6-14 μm.

[0080] In a third aspect, the present application provides a method for spatial transcriptome sequencing, comprising the following steps: obtaining a frozen embedded tissue according to the method for frozen embedding animal vascular tissue described above; slicing the frozen embedded tissue; and performing spatial transcriptome sequencing on the slice.

[0081] In some embodiments, before sequencing, the slice is subjected to quality control, which includes at least one of RNA integrity detection and tissue morphology detection. The RNA integrity detection includes collecting 10-30 slices, extracting RNA, and measuring the RNA RIN value of the tissue (such as by using Agilent 2100 Bioanalyzer). Generally, when the RNA RIN value is above 7, it indicates that the degradation is less and the integrity is better. The tissue morphology test is generally H&E staining, which is used to observe whether the tissue slice is complete in morphology, including whether there are large cracks, bubbles, wrinkles, etc. In some embodiments, the H&E staining procedure is as follows: the slide (or chip) is taken out of the pre-cooled methanol and dried, the tissue is rehydrated, stained with hematoxylin for 1-10 min, washed, differentiated with differentiation solution (such as 1% hydrochloric acid alcohol), washed, stained with eosin for 1-3 min, washed, dried, and covered with glycerol. The tissue is observed under a microscope.

[0082] In some embodiments, before sequencing, the chip is subjected to a pretreatment step, such as a washing step.

[0083] In some embodiments, to better complete the spatial transcriptome experiment, when the slice is subjected to spatial transcriptome sequencing, a plurality of slices are sequentially tiled and combined on the spatial transcriptome sequencing chip, so as to cover enough vascular structures on the surface of the chip. For example, the size of the slice is adjusted by trimming, so as to cover more vascular structures on the surface of the chip. In some embodiments, the number of the slices sequentially tiled and combined on the spatial transcriptome sequencing chip is 2-5, such as 2, 3, 4, or 5. In some embodiments, the tissue slice obtained has an exposed part with a width of less than 0.3 cm and a length of less than 1 cm. A plurality of continuous tissue slices are arranged in parallel to form a combined slice with a length and a width of less than 1 cm, which is completely attached to a 1 cm x 1 cm spatial transcriptome chip without overlapping between the vascular tissues. It can be understood that the size and number of the tissue slices can also be adjusted according to the actual size of the spatial transcriptome chip.

[0084] In some embodiments, after the spatial transcriptome chip is separated from the cover glass and washed, a permeation reagent is added to the surface of the chip for permeation. In some embodiments, the permeation temperature is 37°C and the permeation time is 5-10 minutes. In some embodiments, after permeation, the permeation solution is removed and the chip is washed, followed by reverse transcription to generate cDNA, and amplification such as PCR to construct a cDNA library reflecting the spatial distribution, and sequencing. In some embodiments, after reverse transcription, the liquid on the surface of the spatial transcriptome chip is aspirated, washed, and then reacted with a tissue removal reagent to remove the tissue section on the surface of the chip, followed by release of the cDNA from the chip by a release reagent. In some embodiments, the released cDNA is purified by magnetic beads and then amplified to form a cDNA library. In some embodiments, the cDNA amplification product is subjected to concentration measurement and fragment distribution determination.

[0085] The content of the present application is further described in detail below through specific examples.

[0086] It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application.

[0087] The experimental methods in the following examples, unless otherwise specified, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The materials, reagents, etc. used in the present examples, unless otherwise specified, are reagents and materials obtained from commercial channels.

[0088] The relevant materials involved in the following examples include C57BL / 6J strain mice, S-P Single-Section H&E Staining Kit, Leica fluorescence microscope, Huada STO mics gene expression chip, and Qubit ssDNA HS Reagent from Invitrogen. The remaining conventional materials are shown in Table 1 below.

[0089] Table 1. Materials and manufacturers for experiments

[0090]

[0091]

[0092] Example 1: Mouse aortic vessel separation

[0093] The specific steps are as follows:

[0094] 1. The mouse was euthanized by inhaling carbon dioxide. After spraying the chest with 75% ethanol, the skin and chest wall were carefully cut with scissors to expose the heart.

[0095] 2. To perfuse the vascular system, make a small incision in the right atrium and steadily inject at least 10 mL of pre-chilled PBS (about 1 mL / s) into the apex of the left ventricle using a 21 or 23 G needle. Continue until all organs are white. Good perfusion is indicated when the liver is white.

[0096] 3. Gently cut out the intestines, stomach, spleen, pancreas, and liver with small forceps and fine scissors to better visualize the aorta.

[0097] Note: This step must be done very precisely, as damage to the gastrointestinal tract can cause contamination;

[0098] 4. Cut and remove each lung with scissors. Flush the thoracic cavity with phosphate buffered saline (PBS) using a needleless syringe. Remove excess blood and fluid with sterile gauze.

[0099] 5. Cut the abdominal aorta with fine dissecting forceps to drain the lymph nodes. Be sure not to rupture the cysts. Remove the remaining fat from the surface of each lymph node with dissecting forceps. Then cut out the two kidneys with fine scissors.

[0100] 6. Dissect out the entire aorta (thoracic and abdominal) with fine, curved scissors. Start at the heart and carefully dissect down the esophagus and vertebrae to the iliac bifurcation, making sure that the surrounding fat of the vessel is attached to the aorta.

[0101] 7. Further dissect the aorta under a microscope to remove surrounding structures, cut the branches of the aorta (carotid, subclavian, mesenteric, and renal arteries), and lymph nodes.

[0102] 8. The resulting mouse aorta morphology is shown in Figure 1 .

[0103] Example 2: Mouse aorta vascular embedding

[0104] 1. Different concentrations of OCT perfusion solution test

[0105] Take OCT and PBS mixed in different volume ratios, and add 5% volume percentage of RNase inhibitor to prepare perfusion solutions containing 30% OCT, 50% OCT, 70% OCT, and 100% OCT volume percentages.

[0106] Perfusion the mouse aorta tissue with these perfusion solutions of different concentrations of OCT.

[0107] Crush dry ice into powder and place it in a container box.

[0108] Place the OCT bottle upside down on ice, with the bottle opening buried in crushed ice, and pre-cool for 10 min; after pre-cooling, inject the OCT embedding agent into the embedding box, and the amount of OCT embedding agent added is ≤ 1 / 3 of the volume of the embedding box.

[0109] Confirm the direction of the tissue section, mark the direction, specific location and tissue size on the edge of the embedding box, and put the perfused mouse aortic tissue into the embedding box containing OCT embedding agent, then cover the mouse aortic tissue completely with OCT embedding agent. Ensure that there are no air bubbles around the tissue, and add a layer of lid on top, and put dry ice on the lid to accelerate the freezing speed.

[0110] Immediately place the embedding box on the pre-cooled dry ice until the OCT embedding agent is completely frozen.

[0111] After completing the embedding, perform H&E staining to observe the tissue state, wherein the perfusion fluid of 70% and 100% OCT is too viscous to perform perfusion. The results are shown in Figure 2 As shown, the perfusion fluid of 50% OCT is more full and has no collapse than the perfusion fluid of 30% OCT, so the perfusion fluid of 50% OCT is the most suitable perfusion fluid.

[0112] 2. Embedding of different amounts of vascular tissue and quality testing

[0113] Select the perfusion fluid of 50% OCT to treat the blood vessels; embed 5 vascular tissues in the same OCT embedding block. Complete the preparation of frozen sections and perform H&E staining, and the results are shown in Figure 3 As can be seen from the figure, the 5 vascular tissues are in good condition, the morphology is complete, and the vascular structure is full and has no collapse.

[0114] Based on the above results, A1: 5 vascular tissue sections (5 vascular tissues on one section), 10 vascular tissue sections (5 vascular tissues / section x 2 sections), and 15 vascular tissue sections (5 vascular tissues / section x 3 sections) were cut 30 frozen sections for RNA concentration and fragment analysis to meet the RNA fragment distribution and abundance quality control of spatial transcriptome sequencing experiment, and the results are shown in Table 2. From the results in the table, it can be found that the RNA concentration in the 15 vascular tissue sections with the most vascular tissues is the highest, and the sample quality is the best.

[0115] Table 2. RNA concentration detection results of different vascular quantity sections

[0116] Sample No. Sample Name RNA Concentration A1 5 Vessel Tissue Sections 5.3 ng / μL B1 10 Vessel Tissue Sections 7.6 ng / μL C1 15 Vessel Tissue Sections 10.4 ng / μL

[0117] The steps of RNA quality inspection are as follows:

[0118] (1) Take 30 frozen sections in a 1.5 ml centrifuge tube, add 350 μl of prepared RLT, and mix thoroughly.

[0119] (2) Put the above reagent into a small centrifuge, centrifuge at the maximum speed for 3 min, and recover the supernatant into a new centrifuge tube.

[0120] (3) Add equal volume of 70% ethanol (e.g. 350 μL ethanol to 350 μL supernatant, total volume 700 μL), mix well.

[0121] (4) Transfer the mixed liquid to RNeasy Mini spin column, put it into a small centrifuge, centrifuge at >8000 g for 15 s, retain the column, discard the bottom waste liquid.

[0122] (5) Add 700 μL RW1, centrifuge at >8000 g for 15 s, retain the column, discard the bottom waste liquid.

[0123] (6) Add 500 μL RPE, centrifuge at >8000 g for 15 s, retain the column, discard the bottom waste liquid.

[0124] (7) Add 500 μL RPE, centrifuge at >8000 g for 2 min, retain the column, discard the bottom waste liquid.

[0125] (8) Discard the old collection tube, replace it with a clean 2 mL collection tube, put it into the centrifuge, centrifuge at maximum speed for 1 min.

[0126] (9) Discard the old collection tube, replace it with a clean 1.5 mL centrifuge tube, add 50 μL RNase-free water to the adsorption membrane, centrifuge at >8000 g for 2 min, retain the solution in the collection tube, which is the purified total RNA, quickly place it on ice, and freeze it at -80°C after sampling test.

[0127] (10) If you want to improve the yield, you can do a second redissolution, repeat the above step.

[0128] (11) Test the RNA concentration with nanodrop and record it, analyze the RIN value and fragment distribution of the RNA with Agilent 4200 RNA fragment analyzer, and detect the concentration of the RNA extracted in this experiment with Qubit RNA concentration detection kit. (Appendix Figure 4 )

[0129] (12) Freeze the remaining samples at -80°C for long-term storage.

[0130] The results are as follows Figure 4 , in the three schemes of A1-C1, the RIN value of A1 is only 5.3, the RIN value of B1 is slightly higher than that of A1, and the RIN value of C1 is greater than 7, which meets the requirements of spatial transcriptome experiment. At the same time, the RNA concentration in the tissue sections of 15 blood vessels is the highest, so the embedding scheme of 15 blood vessels is the most suitable scheme.

[0131] Example 3: Mouse blood vessel tissue frozen section

[0132] The specific steps are as follows:

[0133] The cryostat is pre-cooled to -13°C to reach the optimal cryostat temperature.

[0134] Following the aforementioned optimized procedure (50% OCT perfusion solution), the OCT embedding block of the perfused vascular tissue sample was transferred from a -80°C freezer to the microtome for half an hour to equilibrate the temperature. Then, an appropriate amount of OCT embedding agent was added to the sample stage, and the OCT embedding block of the perfused vascular tissue sample was placed on the sample holder and transferred to a quick-freezing stage pre-cooled to -60°C for static placement. Subsequently, it was fixed to the sample head and allowed to warm up to the sample head temperature for 3 minutes.

[0135] Remove excess OCT by first setting the slice thickness to 50 μm and then removing the embedded block until the tissue outline is visible.

[0136] Trim the embedded blocks with a trimmer, and once they meet the requirements, adjust them to 10μm to collect sample slices for RNA quality control.

[0137] When the cut surface is close to the sample to be collected, set the slice thickness to 6μm, 8μm, 10μm, 12μm, and 14μm, lay the slice flat on the quick-freezing stage, and observe the optimal slice thickness and slice quality.

[0138] The results are as follows Figure 5 As shown in the figure, the slice quality of the adjusted 6-14 μm thickness slices meets the requirements of spatial transcriptomics experiments. Considering that the average diameter of vascular cells is about 10 μm, a 10 μm slice thickness is selected as the optimal experimental scheme to ensure that the majority of cells in the slice are monolayer cells, so as to perform spatial omics capture of monolayer cells, complete single-cell spatial resolution analysis, and avoid cross-capture of multilayer cells.

[0139] Example 4: H&E staining of frozen sections of mouse vascular tissue

[0140] The specific steps are as follows:

[0141] The frozen sections obtained by the optimal scheme in Example 3 (50% OCT perfusion solution, 10μm section thickness) were attached to the capture area of ​​the tissue optimization slide. The marking area was marked, and the finger was placed on the back of the capture area of ​​the slide to make the OCT melt quickly, and the section adhered to the slide.

[0142] The slide was then placed in a methanol solution and fixed for 30 minutes.

[0143] Rinse gently with tap water for 1 minute to remove surface methanol.

[0144] Stain with hematoxylin solution for 3 minutes, then gently rinse with tap water for 1 minute.

[0145] Differentiate for 1 min with differentiation solution, rinse gently with tap water for 1 min.

[0146] Stain for 1 min with eosin, rinse gently with tap water for 2 min.

[0147] Place the slide in a slide warmer at 37°C for 1 min.

[0148] Slowly pipette 5 μL of Glycerol onto the center of the tissue, avoiding air bubbles.

[0149] Use forceps to pick up the coverslip, carefully place one end of the coverslip on the edge of the slide while holding the other end, then gradually lower the coverslip onto the slide until it completely covers the slide; after the glycerol has soaked the entire slide, take a 5x background white light photo.

[0150] Example 5: Spatial Transcriptome Sequencing

[0151] The optimal protocol (50% OCT perfusion solution, 10 μm section thickness, 5 blood vessels per block, 3 sections per chip) in the foregoing examples is used as follows:

[0152] (1) Spatial Transcriptome Chip Processing and Pasting

[0153] After the H&E slide is baked, the optimal test condition protocol is used to perform H&E staining experiments and spatial transcriptome chip ssDNA staining experiments to verify whether the combined protocol is suitable for spatial transcriptome experiments.

[0154] Prepare 2 culture dishes, cover the bottom with a suitable size of sealing film, and gently press the film around the edges with the blunt end of the forceps to make it flat and fixed on the bottom of the culture dish, which can prevent the chip from sliding.

[0155] Transfer the chip stored in the TE buffer to a new 24-well plate with forceps, and record the chip number.

[0156] Gently add 400 μL of Nuclease Free Water along the wall of the well, and use a pipette to suck out the liquid from the side without touching the surface of the chip.

[0157] Repeat the previous step.

[0158] Carefully transfer the chip from the well to the tissue paper with forceps to remove the water on the back. One hand holds the chip with forceps and fixes it on the tissue paper, and uses an air tank (MATIN, M-6318) to slowly blow away the excess water from the side of the chip at a horizontal angle of 30-45°.

[0159] Place the chip in the chip oven at 37°C for 1 min. The surface of the chip should be dry and free of any ripples. If ripples are observed, add 400 μL of Nuclease Free Water and pipette the liquid away from the side of the chip without touching the surface.

[0160] Place the frozen section on the right edge of the slide and gently spread the section with a fine brush.

[0161] Gently place the chip on the section using a pair of tweezers. The section should be visible on the surface of the chip. Flip the chip over so that the front is facing up and place it in the oven at 37°C for 3 min.

[0162] (2) Tissue fixation

[0163] Immediately place the chip in -20°C pre-cooled methanol using a pair of tweezers. At this time, check that the microscope is turned on and switched to the FITC channel.

[0164] Remove the chip from the 24-well plate using a pair of tweezers and use a piece of tissue paper to absorb the excess methanol from the back of the chip. Place the chip in a petri dish with parafilm on the bottom. Do not cover the dish and place it in the fume hood for 2-3 min to allow the methanol to evaporate. This completes the tissue fixation.

[0165] (3) Fluorescent staining

[0166] Prepare the fluorescent staining solution by mixing 1.1 μL of Qubit ssDNA dye with 220 μL of 5x SSC solution. Add 100 μL of the fluorescent staining solution to each chip, starting with a drop in each corner and then adding the remaining solution to the center of the chip. Mix the solution to ensure that it covers the entire chip. Allow the chip to stain for 5 min at room temperature in the dark.

[0167] Tilt the petri dish and use a pipette to remove the fluorescent staining solution from one corner of the chip. Try to minimize the amount of liquid on the surface. Add 100 μL of 0.1x SSC to the surface of the chip. Tilt the petri dish and use a pipette to remove the 0.1x SSC from one corner of the chip. Try to minimize the amount of liquid on the surface.

[0168] Transfer the chip to a piece of tissue paper. Use a pair of tweezers to hold the chip with the left hand and an air tank with the right hand. Blow air onto the chip at a 30° angle, starting from one corner and moving across the surface of the chip. Try to avoid blowing too hard.

[0169] Add a drop of water (~1 μL) on the slide, then carefully move the chip onto the slide with forceps, which helps the chip to stick to the slide better.

[0170] Slowly pipette 5 μL of glycerol into the center of the tissue, avoiding the formation of air bubbles.

[0171] Gently pick up the coverslip with forceps, and carefully place one end of the coverslip on the edge of the chip while holding the other end, then gradually lower the coverslip onto the chip until it completely covers the chip. Immediately arrange to take a photo after the glycerol has soaked the entire chip to avoid fluorescence quenching.

[0172] (4) Fluorescence photography

[0173] Create a new folder in the PC connected to the fluorescence microscope, name it with the chip number, and note other relevant information.

[0174] Use the fluorescence microscope, select the epi-fluorescence scanning mode, select the FITC channel, 10x lens, and scan the entire chip to take a full picture.

[0175] Save the entire folder.

[0176] Open the ImageQC software and upload the ssDNA staining picture to determine whether it meets the requirements of spatial transcriptomics. The results, as shown in Figure 6 , indicate that the ssDNA staining picture has a complete tissue state, normal nucleus state, and strong signal, meeting the subsequent spatial transcriptome experiment requirements.

[0177] (5) cDNA quality detection

[0178] Use the Huada Stereo-seq kit and refer to its instructions to complete tissue permeation, reverse transcription, cDNA release recovery, and purification, and detect the fragment distribution of cDNA. The specific steps can be referred to as follows:

[0179] Invert the spatial transcriptome chip that has been photographed in a petri dish, add 5x SSC to completely cover the chip, and permeate for 8 minutes. After the chip and coverslip are completely separated, place the spatial transcriptome chip upright on a dust-free paper, absorb the liquid on the back and around, then use 0.1x SSC to clean the chip surface and discard.

[0180] Slowly add 100 μl of permeation reagent to the chip surface, completely cover the chip surface, and then slowly transfer it to a 37°C oven for permeation for 8 minutes. Absorb the liquid on the surface of the permeated spatial transcriptome chip, use 0.1x SSC to clean it once, add 100 μl of reverse transcription reagent to cover the chip surface, and transfer it to a 42°C oven for reverse transcription reaction for 2 hours.

[0181] The liquid on the surface of the spatial transcriptome chip after reverse transcription is discarded, washed once with 0.1×SSC, transferred into a 24-well plate, 400 μl of tissue removal reagent is added, and transferred into a 55-degree Celsius oven for 10 minutes.

[0182] The 24-well plate is taken out, the spatial transcriptome chip is transferred into a new well, 1 ml of 0.1×SSC reagent is added, the chip surface is gently blown, the tissue is removed to achieve a non-visible effect, then the chip is transferred into a new well, 400 μl of release reagent is added, a sealing film is covered to completely seal it to prevent liquid evaporation, and transferred into a 55-degree Celsius oven for 18 hours.

[0183] The reacted 24-well plate is taken out, the liquid in each reaction well is carefully sucked and transferred into a 1.5 ml EP tube, nuclease-free water is used to clean the reaction well, the original solution and the cleaning solution are mixed, the volume is accurately measured, 0.8 times the volume of magnetic beads is used for purification, cDNA liquid is obtained, a 100 μl PCR system is configured, and PCR is performed, the PCR process is: hot cover 105 degrees Celsius, 95 degrees Celsius for 5 minutes; 98 degrees Celsius for 20 seconds, 58 degrees Celsius for 20 seconds, 72 degrees Celsius for 3 minutes, 18 cycles; 72 degrees Celsius for 5 minutes, 12 degrees Celsius.

[0184] The obtained cDNA amplification liquid is measured for concentration and fragment distribution.

[0185] The results are shown in the following table: Figure 7 As can be seen from the figure, the cDNA fragment distribution is concentrated in about 800-1000 bp, which meets the sequencing requirements.

[0186] From the above examples, it can be seen that the present application can obtain a mouse blood vessel processing overall scheme suitable for spatial transcriptome sequencing. The problems of small volume of mouse blood vessels, small number of cells, low RNA content, and difficult detection are solved; the difficulty of maintaining the shape of the mouse blood vessels is solved; the problem of easy degradation of RNA inside the mouse blood vessel tissue, which reduces the sequencing quality, is solved; the problem of difficulty in slicing mouse blood vessel tissue and the difficulty in obtaining high-quality, morphologically complete, and well-cell-conditioned slices is solved.

[0187] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A method for cryo-embedding animal vascular tissue, characterized by, The method comprises the following steps: replacing blood in the animal with a first buffer solution; stripping the blood vessel and removing surrounding tissues; perfusing the blood vessel with a perfusion solution to obtain a perfused blood vessel, the perfusion solution comprising OCT and a second buffer solution; quick-freezing a plurality of perfused blood vessels embedded in the same OCT embedding block.

2. The method according to claim 1, wherein: the number of blood vessels embedded in the same OCT embedding block is 2-20.

3. The method according to claim 2, wherein: the number of blood vessels embedded in the same OCT embedding block is 2-10.

4. The method according to claim 1, wherein: the perfusion solution further comprises an RNAse inhibitor.

5. The method according to claim 4, wherein: the volume percentage of the RNAse inhibitor in the perfusion solution is 1-10%.

6. The method according to claim 1, wherein: the animal is a rodent.

7. The method according to claim 6, wherein: the animal is a mouse or a rat.

8. The method according to claim 1, wherein: the volume percentage of OCT in the perfusion solution is less than 70%.

9. The method according to claim 8, wherein: the volume percentage of OCT in the perfusion solution is 10-60%.

10. The method according to claim 8, wherein: the volume percentage of OCT in the perfusion solution is 30-60%.

11. The method according to claim 8, wherein: the volume percentage of OCT in the perfusion solution is 45-55%.

12. The method according to claim 1, wherein: the blood vessel is selected from at least one of an artery, a vein and a microvessel.

13. The method according to claim 12, wherein: the artery is selected from at least one of an elastic artery, a muscular artery and a small artery.

14. The method according to claim 12, wherein: the vein is selected from at least one of a large vein, a medium vein and a small vein.

15. The method according to claim 12, wherein: the microvessel is selected from at least one of a continuous microvessel and a permeable microvessel.

16. The method according to claim 13, wherein: the elastic artery is selected from at least one of a thoracic aorta, an abdominal aorta, a subclavian artery and a common carotid artery.

17. A method of sectioning animal vascular tissue, characterized by, The method comprises the following steps: obtaining a frozen embedding tissue according to any one of claims 1-16; slicing the frozen embedding tissue.

18. The method of claim 17, wherein: the thickness of the section is 6-14 microns; and / or the temperature of the sample head during sectioning is -15 to -10 °C.

19. A spatial transcriptome sequencing method, characterized by, comprises the steps of: frozen embedding the tissue according to the method of any one of claims 1 to 16; sectioning the frozen embedded tissue; spatial transcriptome sequencing the section.

20. The method of claim 19, wherein: the sections are sequentially tiled onto a spatial transcriptome sequencing chip.

21. The method of claim 20, wherein: the number of sections sequentially tiled onto the spatial transcriptome sequencing chip is 2-5.

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