WO2022048544A9 - 一种非脱钙骨软骨组织的切片制备方法及其细胞示踪和RNAscope联合测试方法 - Google Patents

一种非脱钙骨软骨组织的切片制备方法及其细胞示踪和RNAscope联合测试方法 Download PDF

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WO2022048544A9
WO2022048544A9 PCT/CN2021/115760 CN2021115760W WO2022048544A9 WO 2022048544 A9 WO2022048544 A9 WO 2022048544A9 CN 2021115760 W CN2021115760 W CN 2021115760W WO 2022048544 A9 WO2022048544 A9 WO 2022048544A9
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osteochondral tissue
rnascope
osteochondral
tissue
kawamoto
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WO2022048544A1 (zh
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杨辉亮
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四川大学华西医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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    • G01N21/64—Fluorescence; Phosphorescence
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    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving

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  • the invention belongs to biological detection technology, and specifically relates to a preparation method of non-decalcified osteochondral tissue slices and a combined test method of osteochondral tissue cell tracing and RNAscope.
  • Fluorescent cell tracking technology has a wide range of applications and can track the development, differentiation and migration of a group or a single cell without affecting the characteristics of the labeled cells themselves and surrounding tissue cells.
  • RNAscope is a novel RNA in situ hybridization technology developed by ACD in the United States. In soft tissue sections, fluorescent cell tracking technology and RNAscope technology can be effectively combined. Thus, the target RNA expression level can be detected in a specific cell population.
  • the present invention uses Kawamoto membrane to ensure the integrity of osteochondral tissue during frozen sectioning Then, the fluorescent cell tracking technology and in situ hybridization technology were combined to observe the fluorescence signal of cell tracking and the specific target RNA signal of RNAsocpe in situ hybridization in the same osteochondral tissue section.
  • a method for preparing a non-decalcified osteochondral tissue slice comprising the following steps:
  • the non-decalcified osteochondral tissue slices are prepared in step (5), they are stored in a refrigerator at -80°C.
  • the present invention also provides a method for combined testing of osteochondral tissue cell tracing and RNAscope, comprising the following steps:
  • the method for constructing osteochondral tissue with cell tracers in step (a) is selected from transgenic labeling, Y chromosome labeling and dye labeling.
  • step (c) the specific process of the RNA in situ hybridization processing described in step (c) is:
  • (c4) Signal amplification the RNAscope detection kit is used to amplify the signal of the target RNA hybridized with the Z-type probe step by step;
  • the amount of the reagent for processing the osteochondral tissue slice is determined according to the size of the osteochondral tissue slice, to ensure that the reagent covers the osteochondral tissue slice on the glass slide.
  • the method for covering the osteochondral tissue slice with the reagent for processing the osteochondral tissue slice comprises the following steps:
  • the detection method in step (d) is observation with an ordinary optical microscope or imaging with a multispectral fluorescence imaging system.
  • the "Kawamoto film” in the present invention refers to the Kawamoto film product produced by SECTION-LAB Co.Ltd.
  • RNAscope pretreatment kit including 1, 2, and 3 reagents
  • Z-type probe used in the RNAscope detection kit
  • RNAscope color reagent used in the RNAscope test of the present invention
  • other reagents belong to mature existing technologies, and commercial reagents can be purchased directly.
  • RNAscope pretreatment kit including 1, 2, and 3 reagents
  • Z-type probe used in the RNAscope test of the present invention
  • RNAscope color reagent used in the RNAscope test of the present invention
  • the present invention can save decalcification time without decalcification, and avoid the destruction and degradation of RNA during decalcification; and frozen section can maintain the morphological integrity of osteochondral tissue under non-decalcification conditions.
  • the slicing method of the present invention is frozen section, which adopts OCT to embed, and does not adopt paraffin to embed, which can avoid the self-fluorescence of paraffin itself under a fluorescence microscope, and reduce the interference to the fluorescence displayed by intracellular fluorescent protein;
  • the osteochondral tissue sections need to be subjected to hydrogen peroxide, acid and alkali treatment.
  • the tapes or the tapes would decrease during the treatment with hydrogen peroxide, protease, ethanol, and other weak acids and bases. Transparency of the membrane, or can lead to delamination of osteochondral tissue sections during treatment with hydrogen peroxide, etc.
  • the present invention selects the Kawamoto membrane to stick and fix the osteochondral tissue, not only can complete and unbroken osteochondral tissue slices be obtained at the slicing stage, but also the bone can be treated with hydrogen peroxide, protease, ethanol, and other weak acids and bases in the subsequent process. During cartilage tissue sectioning, the transparency of Kawamoto's membrane can be maintained and the osteochondral tissue section will not fall off.
  • the present invention can simultaneously realize cell tracking and gene transcription level detection on osteochondral tissue slices. It can be used to study the level of specific RNA in the process of proliferation, differentiation and apoptosis of specific cell populations, so as to understand the role and mechanism of corresponding genes in the process of osteochondral development, injury, repair and regeneration.
  • the applications of the present invention include: 1) research on the development of osteochondral tissue; 2) analysis of the corresponding gene knockout efficiency in osteochondral tissue; 3) research on the effect of gene knockout on the transcription level of other target genes; 4) bone Histopathological analysis of cartilage. Expanded the application scope of cell tracking and RNAscope combined testing technology.
  • reagents are used for soaking in the prior art (eg, experimental manuals of various commercial reagents).
  • the dosage of the reagents is the lowest, that is, the reagents are added dropwise so that the reagents just cover the osteochondral tissue slices.
  • This technical solution greatly reduces the amount of reagents (for example, 700ml of pretreatment reagent 2 dilution solution should be prepared according to the experimental manual) when cooking slices, and has a good processing effect on osteochondral tissue slices, so it can greatly save the cost of testing .
  • Fig. 1 is the process of utilizing Kawamoto membrane to carry out the frozen section of non-decalcified osteochondral tissue
  • Fig. 2 is the signal of the sample after RNAscope 2.5 HD Assay Red Kit processing in embodiment 1 under white light and fluorescence;
  • Fig. 3 is the cell tracing fluorescence signal and RNAscope fluorescence signal observed simultaneously under the fluorescence microscope in Example 1;
  • Fig. 3 is the detection result of the knockout efficiency of target gene in mRNA level in embodiment 2;
  • FIG. 4 shows the pathological changes of osteochondral using the technology of the present invention in Example 3.
  • the preparation method of the osteochondral tissue sections used in Examples 1-3 is as follows:
  • a 6-week old model mouse was used, which was obtained by crossing Shp2fl/fl mice and Agc1-Cre/ER; Rosa26ZsG mice.
  • the mouse genotype is Tg (Agc1-CreER; Shp2 fl/+ ; Rosa26 ZsG ), and the mouse was injected intraperitoneally with tamoxifen within 2 weeks after birth to activate the expression of Cre recombinase, and Cre recombinase is only in Agc1
  • the expression of Cre recombinase can activate the expression of fluorescent protein ZsG, so that all cells expressing Agc1 can express green fluorescent protein ZsG, so that Agc1-positive cells can be traced.
  • RNAscope 2.5 HD Assay Red Kit produced by Advanced Cell Diagnostics was selected for in situ hybridization experiment.
  • the process of the in situ hybridization experiment was performed according to the experimental manual of RNAscope 2.5 HD Assay Red Kit. After soaking in alcohol, the tissue sections were completely air-dried, and wax was drawn on the Kawamoto membrane around the tissue to ensure that the reagents were added dropwise. will not leak.
  • the osteochondral tissue section is processed with the reagent, it is only necessary to cover the osteochondral tissue section with the reagent, that is, the reagent is only within the wax circle.
  • the kit utilizes fast red color development, showing red under white light and red false fluorescence under fluorescence, as shown in Figure 2.
  • the fluorescent signal of cell tracing and the fluorescent signal of RNAscope can be observed at the same time, realizing the application of the combined test of cell tracing and RNAscope in osteochondral tissue sections.
  • Sox9, Ihh and Col10a1 in Figure 3 are early, intermediate and late chondrocyte markers, respectively.
  • cell tracing method and RNAscope were used to detect the knockout efficiency of target genes at the mRNA level on osteochondral tissue sections.
  • Proximal tibiae from 10-week-old Tg (Agc1-CreER; Shp2 fl/+ ; Rosa26 ZsG ) and Tg (Agc1-CreER; Shp2 fl/fl ; Rosa26 ZsG ) model mice were used in this experiment.
  • Tg Agc1-CreER; Shp2 fl/fl ; Rosa26 ZsG
  • in situ hybridization experiments were performed using RNAscope 2.5 HD Assay Red Kit produced by Advanced Cell Diagnostics.
  • This example uses the technical solution of the present invention to study the pathological changes of osteochondral.
  • the proximal tibia of 10-week-old Tg (Ctsk-Cre; Shp2 fl/fl ; Rosa26 ZsG ) model mice was used in this experiment.
  • the chondroma grew on the articular cartilage of the proximal tibia after knocking out SHP2 in Ctsk-positive cells in mice, and the cells of the chondroma originated from Ctsk-positive cells.
  • in situ hybridization experiments were performed using RNAscope 2.5 HD Assay Red Kit produced by Advanced Cell Diagnostics.
  • the non-demineralized osteochondral tissue sections provided by the present invention can withstand the hydrogen peroxide, protease, ethanol, and other weak acids and bases used in the RNAscope test treatment process, and ensure that Kawamoto On the premise that the transparency of the membrane is not reduced and the osteochondral tissue is not peeled off, the combined test of the cell tracing method and the RNAscope is completed.

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Abstract

一种非脱钙骨软骨组织切片的制备方法及骨软骨组织细胞示踪和RNAscope联合测试方法，通过Kawamoto膜保证骨软骨组织在冰冻切片时的完整性，然后联合应用荧光细胞示踪技术和原位杂交技术实现在同一片骨软骨组织切片中同时观察细胞示踪荧光信号和RNAsocpe的原位杂交特异性靶RNA信号。

Description

一种非脱钙骨软骨组织的切片制备方法及其细胞示踪和RNAscope联合测试方法 技术领域
本发明属于生物检测技术，具体涉及一种非脱钙骨软骨组织切片的制备方法及骨软骨组织细胞示踪和RNAscope联合测试方法。
背景技术
荧光细胞示踪技术应用广泛，可在不影响标记细胞本身及其周围组织细胞特性的情况下，追踪一群或单个细胞发育、分化和迁移的过程。RNAscope是一种由美国ACD开发的新型RNA原位杂交技术。在软组织切片中，可以有效联合应用荧光细胞示踪技术和RNAscope技术。从而实现在特定细胞群体中，检测目标RNA表达水平。
但是目前这两种技术的联合应用尚未在骨软骨组织中有效进行。原因有如下几点：(1)脱钙石蜡切片是目前骨软骨组织切片中最常用的方法，但是脱钙会降解骨软骨组织中RNA，从而导致RNA含量降低，影响原位杂交的效果；另外，石蜡会使组织切片自带背景荧光，影响细胞示踪荧光的观察。(2)传统骨软骨组织冰冻切片无法有效保证骨软骨组织的形态完整，且容易掉片，因此无法进行下一步的原位杂交。
发明内容
针对上述现有技术中缺少合适的骨软骨组织切片制备方法导致荧光细胞示踪技术和RNAscope技术联用难以应用于骨软骨组织的问题，本发明通过Kawamoto膜保证骨软骨组织在冰冻切片时的完整性，然后联合应用荧光细胞示踪技术和原位杂交技术实现在同一片骨软骨组织切片中同时观察细胞示踪荧光信号和RNAsocpe的原位杂交特异性靶RNA信号。
一种非脱钙骨软骨组织切片的制备方法，包括如下步骤：
(1)取冷冻及包埋后的待测骨软骨组织，将骨软骨组织块修正；
(2)取Kawamoto膜，修剪成与骨软骨组织块的待切片面对应的大小；
(3)将修剪后的Kawamoto膜的粘性面粘贴在骨软骨组织块待切片的面上；
(4)对骨软骨组织块粘贴Kawamoto膜的一面进行切片，得到粘贴有Kawamoto膜的骨软骨组织切片。
(5)将骨软骨组织切片粘贴有Kawamoto膜的一面贴到载玻片上，固定， 得非脱钙骨软骨组织切片。
优选的，步骤(5)制备得到所述非脱钙骨软骨组织切片后，将其放入-80℃的冰箱中储存。
本发明还提供一种骨软骨组织细胞示踪和RNAscope联合测试的方法，包括如下步骤：
(a)构建具有细胞示踪剂的骨软骨组织；
(b)按照权利要求1所述的制备方法制备得到非脱钙骨软骨组织切片
(c)对固定在载玻片上的骨软骨组织切片进行RNA原位杂交处理；
(d)同时检测骨软骨组织切片中的细胞示踪信号和目标RNA信号。
优选的，步骤(a)中所述构建具有细胞示踪剂的骨软骨组织的方法选自转基因标记、Y染色体标记和染料标记。
优选的，步骤(c)所述RNA原位杂交处理的具体过程为：
(c1)预处理：通过RNAscope预处理试剂盒处理载玻片上固定好的骨软骨组织切片；
(c2)透化：用蛋白酶处理骨软骨组织切片，暴露目标RNA；
(c3)探针杂交：针对靶基因设计RNAscope Z型探针与目标RNA杂交；
(c4)信号放大：采用RNAscope检测试剂盒对与Z型探针杂交后的目标RNA逐级信号放大；
(c5)显色：采用RNAscope显色试剂进行显色。
优选的，步骤(c1)-(c5)中，用于处理骨软骨组织切片的试剂的用量根据骨软骨组织切片的大小确定，确保试剂在载玻片上覆盖骨软骨组织切片。
优选的，步骤(c1)-(c5)中，用于处理骨软骨组织切片的试剂的覆盖骨软骨组织切片的方法包括如下步骤：
(1)将骨软骨组织切片在酒精中浸泡后，完全风干；
(2)在骨软骨组织切片的组织周围的Kawamoto膜上用蜡笔画蜡，用于限定试剂覆盖的范围；
(3)向骨软骨组织切片的组织上滴加试剂，使试剂完全覆盖组织且不泄露出画蜡的范围。
优选的，步骤(d)中检测方法为普通光学显微镜观察或者多光谱荧光成像系统成像。
本发明所述“Kawamoto膜”是指SECTION-LAB Co.Ltd.公司生产的Kawamoto膜产品。
本发明所述在RNAscope测试中所采用的“RNAscope预处理试剂盒(其中包括1、2、3三种试剂)”、“Z型探针”、“RNAscope检测试剂盒”和“RNAscope显色试剂”等试剂均属于成熟的现有技术，可直接购买商业化的试剂。且本申请中对于RNAscope测试中相关试剂的使用未特别说明的部分，均可参照其产品的实验手册的说明进行使用。
采用本发明的技术方案后，取得了如下有益效果：
(1)本发明能够在不进行脱钙，能够节约脱钙时间，以及避免脱钙过程中对RNA的破坏和降解；且在非脱钙条件下冰冻切片能够保持骨软骨组织的形态完整性。
(2)本发明的切片方法为冷冻切片，采用OCT包埋，未采用石蜡包埋，能够避免石蜡自身在荧光显微镜下的自显荧光，减少对细胞内荧光蛋白所示荧光的干扰；
(3)骨软骨组织切片在后续的RNA原位杂交处理中，需要承受过氧化氢、酸和碱的处理。经过对多种胶带和膜的实验，发现如果用其他胶带或膜替代本发明中采用的Kawamoto膜，要么会在过氧化氢、蛋白酶、乙醇，以及其他弱酸和弱碱的处理过程中降低胶带或膜的透明性，要么会在过氧化氢等处理的过程中导致骨软骨组织切片的脱片。
由于本发明选用了Kawamoto膜对骨软骨组织进行粘贴固定，不仅能够在切片阶段得到完整不破碎的骨软骨组织切片，而且在后续用过氧化氢、蛋白酶、乙醇，以及其他弱酸和弱碱处理骨软骨组织切片时还能保持Kawamoto膜的透明性和骨软骨组织切片不脱片。
(4)本发明能够在骨软骨组织切片上同时实现细胞示踪和基因转录水平的检测。能够用于研究特定细胞群体增殖、分化和凋亡过程中特定RNA水平，从而理解相应基因在骨软骨发育、损伤、修复和再生过程中的作用和机制。具体的，本发明的应用包括：1)骨软骨组织发育研究；2)骨软骨组织中相应基因敲除效率分析；3)基因敲除后对其他目标基因转录水平的影响的研究；4)骨软骨组织病理分析。拓展了细胞示踪和RNAscope联合测试技术的应用范围。
(5)在RNAscope对骨软骨组织切片的处理过程中，现有技术(例如各种商业化试剂的实验手册)中均采用较大量的试剂进行浸泡。而本发明的优选方案中，试剂用量均采用最低量，即，滴加试剂，使试剂刚好覆盖骨软骨组织切片。该技术方案大大降低了试剂的用量(比如煮片时预处理试剂2稀 释液依照实验手册上所述需准备700ml)，且对骨软骨组织切片具备良好的处理效果，因而能够大大节约测试的成本。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为利用Kawamoto膜进行非脱钙骨软骨组织冰冻切片的过程；
图2为实施例1中RNAscope 2.5 HD Assay Red Kit处理后的样品在白光和荧光下的信号；
图3为实施例1中在荧光显微镜下同时观察到的细胞示踪荧光信号和RNAscope荧光信号；
图3为实施例2中靶基因在mRNA水平中敲除的效率的检测结果；
图4为实施例3中利用本发明技术研究骨软骨病理变化。
具体实施方式
以下实施例均在中国博士后科学基金面上项目(2019M653417)和四川省科技计划(2020YJ0025)资助下完成:
实施例1-3所用的骨软骨组织切片的制备方法如下：
(1)取冷冻及包埋后的待测骨软骨组织，修成骨软骨组织块；
(2)取Kawamoto膜，修剪成与骨软骨组织块的待切片面对应的大小；
(3)将修剪后的Kawamoto膜的粘性面粘贴在骨软骨组织块待切片的面上；
(4)对骨软骨组织块粘贴Kawamoto膜的一面进行切片，得到粘贴有Kawamoto膜的的骨软骨组织切片；
(5)将骨软骨组织切片中粘贴有骨软骨组织的Kawamoto膜贴到载玻片上，储存在-80℃冰箱内，供后期免疫组化、原位杂交等分析使用。
上述制备流程如图1所示。
实施例1
本实施例中使用了6周大模式小鼠，该模式小鼠用Shp2fl/fl小鼠与Agc1-Cre/ER；Rosa26ZsG小鼠杂交获得。该小鼠基因型为， Tg(Agc1-CreER；Shp2 fl/+；Rosa26 ZsG),该小鼠在出生后2周内腹腔注射他莫昔芬激活Cre重组酶的表达，Cre重组酶仅在Agc1启动子作用下才能表达，Cre重组酶的表达能够激活荧光蛋白ZsG的表达，从而实现Agc1表达的细胞均有绿色荧光蛋白ZsG的表达，从而可以示踪Agc1阳性的细胞。
在制备得到该模式小鼠的的胫骨近端的粘贴有Kawamoto膜的骨软骨组织切片后，本实施例中选择Advanced Cell Diagnostics公司生产的RNAscope 2.5 HD Assay Red Kit进行原位杂交杂交实验。原位杂交杂交实验的过程按照RNAscope 2.5 HD Assay Red Kit的实验手册进行操作，其中在浸泡酒精之后，待组织切片完全风干，在组织周围的Kawamoto膜上用蜡笔画蜡，保证后续滴加试剂时不会泄露。每次用试剂对骨软骨组织切片进行处理时，仅需要用试剂对骨软骨组织切片进行覆盖即可，即试剂仅在蜡圈之内。该试剂盒利用快红显色，在白光下呈现红色，在荧光下呈现红色假荧光，如图2所示。在荧光显微镜下可同时观察细胞示踪荧光信号和RNAscope荧光信号，实现了细胞示踪和RNAscope联合测试在骨软骨组织切片中的应用。图3中Sox9、Ihh和Col10a1分别是早、中和晚期软骨细胞标记。
实施例2
本实施例在骨软骨组织切片上利用细胞示踪法和RNAscope检测靶基因在mRNA水平上敲除的效率。本实验使用了10周大Tg(Agc1-CreER；Shp2 fl/+；Rosa26 ZsG)和Tg(Agc1-CreER；Shp2 fl/fl；Rosa26 ZsG)模式小鼠的胫骨近端。在制备得到粘贴有Kawamoto膜的骨软骨组织切片后，采用Advanced Cell Diagnostics公司生产的RNAscope 2.5 HD Assay Red Kit进行原位杂交杂交实验。原位杂交杂交实验的过程按照RNAscope 2.5 HD Assay Red Kit的实验手册进行操作，且每次用试剂对骨软骨组织切片进行处理时，仅需要用试剂对骨软骨组织切片进行覆盖即可。检测结果如图4所示。计算生长板中间区域100个绿色细胞中的红点数量，发现红点显著降低77％，p<0.001(n＝5)。
实施例3
本实施例利用本发明的技术方案研究骨软骨病理变化。本实验使用了10周大Tg(Ctsk-Cre；Shp2 fl/fl；Rosa26 ZsG)模式小鼠的胫骨近端。小鼠在Ctsk阳性细胞中敲除SHP2后胫骨近端关节软骨上长出的软骨瘤，该软骨瘤的细胞起源于Ctsk阳性的细胞。在制备得到粘贴有Kawamoto膜的骨软骨组织切片后，采用Advanced Cell Diagnostics公司生产的RNAscope 2.5 HD Assay Red Kit 进行原位杂交杂交实验。原位杂交杂交实验的过程按照RNAscope 2.5 HD Assay Red Kit的实验手册进行操作，且每次用试剂对骨软骨组织切片进行处理时，仅需要用试剂对骨软骨组织切片进行覆盖即可。检测结果如图5所示。图中能够观察到该软骨瘤不同部位的组织表达软骨细胞早(Sox9)、中(Ihh、Col2a1)和晚(Col10a1)期的标记。
从实施例1-3可以看到，本发明提供的非脱钙骨软骨组织切片能够承受RNAscope测试的处理过程中所采用的过氧化氢、蛋白酶、乙醇，以及其他弱酸和弱碱，在保证Kawamoto膜透明性不降低、骨软骨组织不脱片的前提下，完成细胞示踪法和RNAscope的联合测试。

Claims (7)

  1. 一种非脱钙骨软骨组织切片的制备方法，其特征在于，包括如下步骤：
    (1)取冷冻及包埋后的待测骨软骨组织，将骨软骨组织块修正；
    (2)取Kawamoto膜，修剪成与骨软骨组织块的待切片面对应的大小；
    (3)将修剪后的Kawamoto膜的粘性面粘贴在骨软骨组织块待切片的面上；
    (4)对骨软骨组织块粘贴Kawamoto膜的一面进行切片，得到粘贴有Kawamoto膜的骨软骨组织切片；
    (5)将骨软骨组织切片粘贴有Kawamoto膜的一面贴到载玻片上，固定，得非脱钙骨软骨组织切片。
  2. 一种骨软骨组织细胞示踪和RNAscope联合测试的方法，其特征在于，包括如下步骤：
    (a)构建具有细胞示踪剂的骨软骨组织；
    (b)按照权利要求1所述的制备方法制备得到非脱钙骨软骨组织切片
    (c)对固定在载玻片上的骨软骨组织切片进行RNA原位杂交处理；
    (d)同时检测骨软骨组织切片中的细胞示踪信号和目标RNA信号。
  3. 按照权利要求2所述的一种骨软骨组织细胞示踪和RNAscope联合测试的方法，其特征在于：步骤(a)中所述构建具有细胞示踪剂的骨软骨组织的方法选自转基因标记、Y染色体标记和染料标记。
  4. 按照权利要求2所述的一种骨软骨组织细胞示踪和RNAscope联合测试的方法，其特征在于，步骤(c)所述RNA原位杂交处理的具体过程为：
    (c1)预处理：通过RNAscope预处理试剂盒处理载玻片上固定好的骨软骨组织切片；
    (c2)透化：用蛋白酶处理骨软骨组织切片，暴露目标RNA；
    (c3)探针杂交：针对靶基因设计RNAscope Z型探针与目标RNA杂交；
    (c4)信号放大：采用RNAscope检测试剂盒对与Z型探针杂交后的目标RNA逐级信号放大；
    (c5)显色：采用RNAscope显色试剂进行显色。
  5. 按照权利要求4所述的一种骨软骨组织细胞示踪和RNAscope联合测试的方法，其特征在于：所述步骤(c1)-(c5)中，用于处理骨软骨组织切片的试剂的用量根据骨软骨组织切片的大小确定，确保试剂在载玻片上覆盖骨软骨组织切片。
  6. 按照权利要求5所述的一种骨软骨组织细胞示踪和RNAscope联合测试的方法，其特征在于：所述步骤(c1)-(c5)中，用于处理骨软骨组织切片的试剂的覆盖骨软骨组织切片的方法包括如下步骤：
    (1)将骨软骨组织切片在酒精中浸泡后，完全风干；
    (2)在骨软骨组织切片的组织周围的Kawamoto膜上用蜡笔画蜡，用于限定试剂覆盖的范围；
    (3)向骨软骨组织切片的组织上滴加试剂，使试剂完全覆盖组织且不泄露出画蜡的范围。
  7. 按照权利要求2所述的一种骨软骨组织细胞示踪和RNAscope联合测试的方法，其特征在于：步骤(d)中检测方法为普通光学显微镜观察或者多光谱荧光成像系统成像。
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