CN112014187A - 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 PDFInfo
- Publication number
- CN112014187A CN112014187A CN202010930339.7A CN202010930339A CN112014187A CN 112014187 A CN112014187 A CN 112014187A CN 202010930339 A CN202010930339 A CN 202010930339A CN 112014187 A CN112014187 A CN 112014187A
- Authority
- CN
- China
- Prior art keywords
- osteochondral
- tissue
- osteochondral tissue
- rnascope
- kawamoto
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000012360 testing method Methods 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 210000001519 tissue Anatomy 0.000 claims abstract description 102
- 239000012528 membrane Substances 0.000 claims abstract description 29
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 23
- 238000007901 in situ hybridization Methods 0.000 claims abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 210000000845 cartilage Anatomy 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 38
- 238000000034 method Methods 0.000 claims description 32
- 239000003153 chemical reaction reagent Substances 0.000 claims description 31
- 230000008569 process Effects 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 239000011521 glass Substances 0.000 claims description 9
- 239000000523 sample Substances 0.000 claims description 8
- 238000011161 development Methods 0.000 claims description 7
- 239000003550 marker Substances 0.000 claims description 6
- 108091005804 Peptidases Proteins 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000000700 radioactive tracer Substances 0.000 claims description 4
- 239000000853 adhesive Substances 0.000 claims description 3
- 230000001070 adhesive effect Effects 0.000 claims description 3
- 238000010008 shearing Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 108700019146 Transgenes Proteins 0.000 claims description 2
- 210000002593 Y chromosome Anatomy 0.000 claims description 2
- 238000007605 air drying Methods 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 230000001413 cellular effect Effects 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 238000000799 fluorescence microscopy Methods 0.000 claims description 2
- 238000009396 hybridization Methods 0.000 claims description 2
- 238000003384 imaging method Methods 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 230000003287 optical effect Effects 0.000 claims description 2
- 238000010422 painting Methods 0.000 claims description 2
- 230000008823 permeabilization Effects 0.000 claims description 2
- 210000003321 cartilage cell Anatomy 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 15
- 238000003209 gene knockout Methods 0.000 abstract description 4
- 238000013518 transcription Methods 0.000 abstract description 3
- 230000035897 transcription Effects 0.000 abstract description 3
- 238000010827 pathological analysis Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 238000010998 test method Methods 0.000 abstract description 2
- 230000025366 tissue development Effects 0.000 abstract description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 14
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 238000003556 assay Methods 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000012188 paraffin wax Substances 0.000 description 4
- 239000001993 wax Substances 0.000 description 4
- 101150091111 ACAN gene Proteins 0.000 description 3
- 201000005262 Chondroma Diseases 0.000 description 3
- 108010051219 Cre recombinase Proteins 0.000 description 3
- 239000002313 adhesive film Substances 0.000 description 3
- 239000002390 adhesive tape Substances 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 210000002303 tibia Anatomy 0.000 description 3
- 101150011252 CTSK gene Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 101150106167 SOX9 gene Proteins 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- 241000212749 Zesius chrysomallus Species 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 210000001612 chondrocyte Anatomy 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 101150082216 COL2A1 gene Proteins 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 102100033019 Tyrosine-protein phosphatase non-receptor type 11 Human genes 0.000 description 1
- 101710116241 Tyrosine-protein phosphatase non-receptor type 11 Proteins 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- WLDHEUZGFKACJH-UHFFFAOYSA-K amaranth Chemical compound [Na+].[Na+].[Na+].C12=CC=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(O)=C1N=NC1=CC=C(S([O-])(=O)=O)C2=CC=CC=C12 WLDHEUZGFKACJH-UHFFFAOYSA-K 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000001188 articular cartilage Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000003197 gene knockdown Methods 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 210000004349 growth plate Anatomy 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- 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
- G01N1/42—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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
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。针对现有技术中由于非脱钙骨软骨组织切片难以保持完整,导致细胞示踪和RNAscope联合测试的技术无法在骨软骨组织切片中应用的问题。本发明的技术方案是: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。本发明拓展了细胞示踪和RNAscope联合测试技术的应用范围,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。
Description
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 2.5HD 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4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5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;Shp2fl/+;Rosa26ZsG),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RNAscope2.5HD 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 2.5HD 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;Shp2fl/+;Rosa26ZsG)和Tg(Agc1-CreER;Shp2fl/fl;Rosa26ZsG)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 2.5HD Assay RedKit进行原位杂交杂交实验。原位杂交杂交实验的过程按照RNAscope 2.5HD Assay RedKit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;Shp2fl/fl;Rosa26ZsG)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 2.5HD 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 2.5HD 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。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010930339.7A CN112014187A (zh) | 2020-09-07 | 2020-09-07 | 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 |
PCT/CN2021/115760 WO2022048544A1 (zh) | 2020-09-07 | 2021-08-31 | 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 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010930339.7A CN112014187A (zh) | 2020-09-07 | 2020-09-07 | 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 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112014187A true CN112014187A (zh) | 2020-12-01 |
Family
ID=73516560
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010930339.7A Pending CN112014187A (zh) | 2020-09-07 | 2020-09-07 | 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 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN112014187A (zh) |
WO (1) | WO2022048544A1 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022048544A1 (zh) * | 2020-09-07 | 2022-03-10 | 四川大学华西医院 | 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 |
WO2023236033A1 (zh) * | 2022-06-07 | 2023-12-14 | 中国科学院深圳先进技术研究院 | 一种转基因动物模型及其构建方法和应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6573043B1 (en) * | 1998-10-07 | 2003-06-03 | Genentech, Inc. | Tissue analysis and kits therefor |
EP2500439A1 (en) * | 2005-06-20 | 2012-09-19 | Advanced Cell Diagnostics, Inc. | Methods of detecting nucleic acids in individual cells and of identifying rare cells from large heterogeneous cell populations |
CN103476948A (zh) * | 2011-01-28 | 2013-12-25 | 领先细胞医疗诊断有限公司 | 用于测定头颈癌和宫颈病变中的hpv状态的rnascope* hpv测定法 |
US20140011202A1 (en) * | 2012-07-03 | 2014-01-09 | The University Of Akron | Decalcification Solution with Preservation of RNA |
CN106636318A (zh) * | 2015-10-30 | 2017-05-10 | 益善生物技术股份有限公司 | 核酸信号放大检测试剂盒 |
CN111504742A (zh) * | 2020-04-24 | 2020-08-07 | 重庆市食品药品检验检测研究院 | 一种基于激光显微切割技术的骨组织细胞收集方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112014187A (zh) * | 2020-09-07 | 2020-12-01 | 四川大学华西医院 | 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 |
-
2020
- 2020-09-07 CN CN202010930339.7A patent/CN112014187A/zh active Pending
-
2021
- 2021-08-31 WO PCT/CN2021/115760 patent/WO2022048544A1/zh active Application Filing
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6573043B1 (en) * | 1998-10-07 | 2003-06-03 | Genentech, Inc. | Tissue analysis and kits therefor |
EP2500439A1 (en) * | 2005-06-20 | 2012-09-19 | Advanced Cell Diagnostics, Inc. | Methods of detecting nucleic acids in individual cells and of identifying rare cells from large heterogeneous cell populations |
CN103476948A (zh) * | 2011-01-28 | 2013-12-25 | 领先细胞医疗诊断有限公司 | 用于测定头颈癌和宫颈病变中的hpv状态的rnascope* hpv测定法 |
US20140011202A1 (en) * | 2012-07-03 | 2014-01-09 | The University Of Akron | Decalcification Solution with Preservation of RNA |
CN106636318A (zh) * | 2015-10-30 | 2017-05-10 | 益善生物技术股份有限公司 | 核酸信号放大检测试剂盒 |
CN111504742A (zh) * | 2020-04-24 | 2020-08-07 | 重庆市食品药品检验检测研究院 | 一种基于激光显微切割技术的骨组织细胞收集方法 |
Non-Patent Citations (4)
Title |
---|
KAWAMOTO 等: "《Preparation of Thin Frozen Sections from Nonfixed and Undecalcified Hard Tissues Using Kawamot"s Film Method (2012)》", 《METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.)》, vol. 1130, 31 December 2014 (2014-12-31), pages 152 - 156 * |
卞修武 等: "《分子病理与精准诊断》", 31 January 2020, 上海交通大学出版社, pages: 051 - 052 * |
常威 等: "《肿瘤常见疾病诊治精要》", 30 June 2018, 湖北科学技术出版社, pages: 54 * |
杨海山 等: "《影像诊断新技术》", 31 August 2006, 吉林科学技术出版社, pages: 348 - 349 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022048544A1 (zh) * | 2020-09-07 | 2022-03-10 | 四川大学华西医院 | 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 |
WO2023236033A1 (zh) * | 2022-06-07 | 2023-12-14 | 中国科学院深圳先进技术研究院 | 一种转基因动物模型及其构建方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
WO2022048544A9 (zh) | 2022-04-07 |
WO2022048544A1 (zh) | 2022-03-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20180171292A1 (en) | Method of producing retinal pigment epithelial cell sheet | |
CN112014187A (zh) | 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 | |
EP3395942A1 (en) | Bi- or multi-differentiated organoid | |
US9404908B2 (en) | Non-invasive imaging to the blastocyst stage for the detection of human embryonic aneuploidy | |
US20100267571A1 (en) | Novel method for specimen preparation, which ensures preservation of tissue morphology and nucleic acid quality | |
CN109187118B (zh) | 一种硬组织病理切片展片的方法及装置 | |
JP2011512146A (ja) | 上皮細胞のクローン培養のための系および方法 | |
CN102559909A (zh) | 一种悬钩子属植物中期染色体荧光原位杂交方法 | |
Zeng et al. | Comparison of global gene expression between porcine testis tissue xenografts and porcine testis in situ | |
CN105039569B (zh) | 一种相互易位染色体的断点分析方法 | |
CN110823655A (zh) | 一种激光显微切割获取抗体标记组织单细胞的方法 | |
CN104862316A (zh) | 具有显著促进牛前脂肪细胞增殖生物功能的miRNA-2400 | |
RU2675376C1 (ru) | Способ количественного анализа клеточной составляющей скаффолда | |
Bath | Human corneal epithelial subpopulations: oxygen dependent ex vivo expansion and transcriptional profiling | |
WO2021182633A1 (ja) | 細胞培養物、細胞培養物の評価方法、細胞培養物の製造方法、及び軟骨様組織形成特性評価用マーカー | |
JP5388233B2 (ja) | 再生軟骨の軟骨特性を評価する方法 | |
CN113930388A (zh) | 一种化学品胚胎毒性预测模型及其建立方法 | |
US20200390825A1 (en) | Pluripotent stem cell-directed model of autosomal dominant polycystic kidney disease for disease mechanism and drug discovery | |
Yang et al. | Benchmark for establishment of organoids from gastrointestinal epithelium and cancer based on available consumables and reagents | |
RU2407278C1 (ru) | Способ определения жизнеспособности озимой культуры | |
WO2022071458A1 (ja) | 老化細胞判定システム | |
WO2022071459A1 (ja) | 老化細胞判定システム | |
CN110082180A (zh) | 一种基于新鲜癌组织贴片法制备检测标本的方法 | |
JP4896516B2 (ja) | 組織切片チップとこれを用いた解析データベースの作成方法および細胞または組織の特性診断システム | |
CN113866199A (zh) | 一种鱼类脂肪组织沉积部位和脂肪细胞特征的鉴定方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201201 |