Microsoft Word - cm0801.doc

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1 Sample Preparation for Confocal Microscpic Examination 송형근충북대학교의과대학 목 차 I. 개관 II. PROTOCOLS Method I. Staining suspension cells to slide Method II. Staining frozen tissue sections Method III. Staining growing adherent cells directly on tissue culture dishes Method VI. FISH(fluorescence in situ hybridization) Method V. PRINS(primed in situ synthesis) Labeling III. 참고문헌 (FISH, PRINS)

2 I. 개관 Confocal microscope도기본적으로는현미경이므로현미경관찰을위한시료의제작과정을거치게된다. 관찰대상은크게배양용기내에서부유상태로있는세포들, 부착상태에있는세포들그리고동결절편및파라핀포매조직과염색체등으로나눌수있겠다. 관찰의대상이되는구조물은세포표면항원, 세포내 cytoskeleton(actin 등 ), 세포내단백, 세포내구조물 (mitochondria 등 ), 염색체내단백및염색체내유전자등이되겠고, 관찰대상이되는구조물에대한항체특히형광화된적접항체가있는경우에관찰이가능하다. 최근에는이중형광염색을통해세포내에서특정구조물과특정단백의생성부위를관찰할수있게되었고, 특정단백의이동경로에대한관찰도가능하게되었다. 실제염색에서문제가되는부분은형광을사용하므로서발생되는높은 background를어떻게낮출것인가? 와세포내구조물을관찰하는경우효과적인투과성 (permeabilization) 을어떻게확보할것인가? 하는점이다. 비특이적반응 (nonspecific reaction) 을줄이기위한고전적인방법은적합한 blocking agent의사용이되겠다. 특히동결절편조직을사용하는경우높은배경형광염색때문에어려움을겪게되는데이는최적의동결절편획득및보관 ( 초기박절시충분한 air dry필요 ) 상태가일차적인문제가되며조직과같은종의 sera를 blocking agent로사용하는것이비특이반응을낮출수있는요령이되겠다. 투과성을유도하는제재로 0.1% Triton-X 100과 1% saponin이가장흔히사용되고있는바세포나조직에따라비교사용으로인한선택이필요한것으로생각된다. 염색체에대한형광염색은 FISH(fluorescence in situ hybridization), PRINS(Primed in situ synthesis) 의두가지방법에있는데전자는형광화된 probe를직접 in situ에이용하는방법이고후자는특정유전자의 primer를이용하여 nucleotide의생성을유도하는방법이다. 두방법모두염색체의 DNA를대상으로시행되는바염색체표본의최적조건을갖추는것이좋은결과를위한선별조건이되겠다. 염색체는수확 (harvest), 고정, drying(aging) 과정이모두중요하며특히충분한 aging은결과를좌우하는결정적인요소이다. 각방법들에대한구체적인 protocol을간략히요약하였다. Ⅱ. PROTOCOLS Method I. Staining suspension cells to slide A. PREPARING Attaching suspension cells to slides using a cytocentrifuge. 1. 1x PBS 로 cell 을 washing 후 1x10 6 / ml ~ 2x10 6 / ml로 resuspension 2. Cytocentrifuge; slide 당 cell suspension 0.1~0.5 ml을 1200rpm 에서 5 분동안 spin 한다.

3 B. FIXING fixing 방법은여러가지로필요에따라적절한방법을선택한다. Fixing attached cells in paraformaldehyde. 1. Fresh 4% paraformaldehyde soln. in PBS 준비한다. 2. Slide 를 PBS 로주의하여 rinse 한다. 3. Specimen 이마르지않도록 PBS 를제거한다. 4. 4% paraformaldehyde solution 에담가실온에서 5 분동안 incubation. 5. PBS 로 2 회 washing % triton X-100 또는 NP-40 in PBS 를실온에서 10min 동안처리하여 permeabilize. 7. 1x PBS 로 4 회정도부드럽게 rinse. C. STAINING Antibody binding and detection 1. Slide 에 primary Ab 를첨가하여실온에서최소 30min ~1 시간정도반응시킨다. 2. PBS 로 3 회 washing 3. Fluorochrome-conjcgated 2' Ab 를첨가하여실온에서최소 20min 정도반응시킨다 (1 시간이상은피함. ). 4. PBS 로 3 회 washing. 5. Gelvatol 이나 Mowiol 이첨가된 glycerol 로 mount 한다. Fluorescence detection 할경우 DABCO(1.4-diazobicyclo-[2,2,2]-octane) 을 2.5% 첨가하여사용한다. 6. 형광현미경이나 confocal microscope 으로관찰한다. Method II. Staining frozen tissue sections. A. TISSUE PREPARATION AND SECTIONING 1. 손상되지않은 tissue 를 1cm x 0.4cm 정도의크기로준비한다음 dry ice 를이용하여얼린후 cryostat section 을할수있도록 mount 한다. 2. Sectioning 전 slide 는 1% gelatin 으로 coating 하여준비한다. 3. Tissue 는 5-10 μm로 section 하여 coated slide 에부착한다. 4. Slide 는건조시켜, acetone: methanol(50vol/50vol) 에 10min 간고정한후 air-dry ( 충분히 ) B. BINDING ANTIBODIES TO TISSUE SECTIONS 1. Blocking; 10% human sera 로 15-30min 동안 humidified chamber 에서반응시킨다. 2. Blocking sera 를제거후 primary Antibody solution(diluted in PBS containg 3% BSA) 을약 μg / slide 로 adding 후실온에서최소 30 분동안반응시킨다. (sensitivity 를높이려면, 4, 0/N)

4 3. PBS 로 3 회 washing 4. 2 Ab solution(3% BSA/PBS) 을 adding 후실온에서최소 20 분동안반응시킨다. 5. PBS 로 3 회 washing Method III. Staining growing adherent cells directly on tissue culture dishes. A. Cytoplasmic granule(factor-viii), Membrane Protein (PECAM-1), Membrane recetor(integrin β 1) 의염색방법 1. Medium 을버리고 PBS 300 μl / well 을처리하여 3 번 washing % Triton X-100 이들어있는 2% PFA 300 μl / well 을처리하여실온에서 10 분간 fixation M glycine-pbs 300 μl / well 을처리하여 3 번 washing % normal goat serum 300 μl / well 을처리하여 37 에서 30 분간 incubation 5. Goat serum 을버리고, primary antibody (FVIII, PECAM, or Integrin β1) 을 1:100 으로희석하여 300 μl / well 씩처리한후, 37 에서 1 시간배양. 6. PBS 300 μl / well 을처리하여 shaker 에서 10 분씩 3 번 washing. 7. Secondary antibody 을아래와같이준비하여 300 μl / well 씩처리한후, 37 에서 30 분 incubation. Secondary antibody: - FITC - 25ug/ml RNase - 0.5mg/ml PI - 10ug/ml % Triton X-100 이들어있는 PBS 300 μl / well 을처리하여 shaker 에서 10 분씩 3 번 washing. 9. PBS(pH 8.0) 300 μl / well 을처리하여 shaker 에서 10 분 washing. 10. Slide glass 위에 gelvatol 4 μl drop 11. Cover-slip 을 gelvatol 위에붙임. 12. Fluorescence microscope 으로관찰. B. Actin staining 1. Medium 을버리고 PBS 300 μl / well 을처리하여 3 번 washing % Triton X-100 이들어있는 2% PFA 300 μl / well 을처리하여실온에서 10 분간 fixation M glycine-pbs 300 μl / well 을처리하여 3 번 washing. 4. Phalloidin(100ng/ml) 을처리하여 37 에서 30 분간배양 % Triton X-100 이포함되어있는 PBS 300 μl / well 을처리하여 shaker 에서 10 분씩 3 번 washing. 6. PBS 로 10 분간 washing.

5 7. Slide glass 위에 gelvatol 4 μl drop 8. Cover-slip 을 gelvatold 위에붙임. 9. Fluorescence microscope 으로관찰. 시약회사명 Cat. No 비고 Phalloidin-TRITC Sigma P ug/ml C. Mitochondria staining 1. Medium 을버리고 PBS 300 μl / well 을처리하여 3 번 washing 2. Mito Tracker FM(200 nm) 300 μl / well 을처리하여 37 에서 30 분배양 3. PBS 300 μl / well 을처리하여 shaker 에서 10 분씩 3 번 washing. 4. 4% PFA 300 μl / well 을처리하여 37 에서 15 분간고정. 5. PBS-glycine 300 μl / well 을처리하여 3 번 washing % Triton X-100 이포함되어있는 PBS 300 μl / well 을처리하여 shaker 에서 10 분씩 3 번 washing. 7. PBS 300 μl / well 을처리하여 shaker 에서 10 분 washing. 8. Slide glass 위에 gelvatol 4 μl drop. 9. Cover-slip 을 gelvatol 위에붙임. 10. Fluorescence microscope 으로관찰. 시약회사명 Cat. No 비고 MitoTracker Green FM Molecular Probe M uM Method VI. FISH(fluorescence in situ hybridization) A. FOR CHROMOSOME DENATURATION 1. Chromosome harvest 2. Chromomsome suspension on slide hardening(overnight or 3-4 hrs, 65 dry oven) 3. Washing : 1X PBS, 3times, each others 5min 4. Dehydrate: 70%, 95%, 100% EtOH, each others 5min 5. RNase(100ug/ml), 37, 1hr 6. Washing : 2X ssc, 3times, each others 5min 7. Dehydrate: 70%, 95%, 100% EtOH, each others 5min 8. Denaturation : 70% formamide / 2X ssc, 70, 2min

6 9. Cold 70%, 95%, 100% EtOH, each others 5min B. FOR PROBE 1. salmon sperm DNA(small fragmented DNA) 를끓는물에서 ( ) denaturate for 10min 2. denatured ssdna 는 -70 에서재빨리식힌다. 3. probe reaction mixture probe xλ (biotinylated Ab or chromosome) denatured ss DNA 4λ (non specific binding 방지위해 ) t RNA 2λ 4M LiCl 20λ ( 냉장보관중 ) DW to 100λ (DW를 adding함으로써 total 100λ 가되야함 ) Ethanol 250λ ( total 100λ 중 2.5배가되야함 )(-20 보관 ) salt 와 DNA가엉기는것을볼수있음 에서 30min incubation 5. centrifuge for 15min at 10,000rpm (10min at 15,000rpm) 6. 상층액은버리고 colded 70% alcohol(-70 or -20 ) 로 washing 후 dehydration (eppendorff tube 벽에붙은 salt(licl) 를제거 ) 7. incubator 나 dry oven(35-40 ) 에서말린다. 8. formamide 5λ (denaturing reagent) 를넣어실온에서방치 9. denature for 10min at 75 꺼냄 8) add equal volume(5λ ) hybrid buffer 20x SSC 1λ 5% BSA 2λ 50% Dextran sulfate 2λ buffer 넣고잘 mix. 거품은 down 10. Hybridization: chromosome on slide + probe incubate at 37 in humid incubator overnight C. DETECTION 1. washing (the most important step) 1 in 50% formamide / 2x SSC 3times for 5min each at 42 2 in 0.1x SSC 3times for 5min each at 60 (non-binding & non-specific binding 제거 )

7 2. Detection (humidified condition) 1 blocking step : incubate with blocking sol(3% BSA / 4x SSC -20 ) for 30min at 37 2 incubate with FITC-conjugated avidine DN(2.5λ /500 μl 1BSA) for 30min at 37 (80λ ) 3 washing in 4x SSC / 0.1% Tween20 3times for 5min each at 42 4 incubate with biotinylated anti-avidin(6 μl /500 μl 1BSA) for 30min at 37 5 washing in 4x SSC / 0.1% Tween20 3times for 5min each at 42 6 incubate with FITC-conjugated avidin DN(2.5 μl /500 μl 1BSA) for 30min at 37 7 washing in 4x SSC / 0.1% Tween20 3times for 5min each at countstaining mounting DABCO 1ml PI 1.25μl PPD 5μl DABCO 1ml에 PI 와 PPD를넣어 voltexing Method V. PRINS(primed in situ synthesis) Labeling A. FOR CHROMOSOME DENATURATION 1. Chromosome harvest. 2. Chromomsome suspension on slide hardening(overnight or 3-4hrs, 65 dry oven) 3. Washing : 1X PBS, 3times, each others 5min 4. Dehydrate: 70%, 95%, 100% EtOH, each others 5min 5. RNase(100ug/ml), 37, 1hr 6. Washing : 2x ssc, 3times, each others 5min 7. Dehydrate: 70%, 95%, 100% EtOH, each others 5min 8. Denaturation : 70% formamide / 2x ssc, 70, 2min 9. Cold 70%, 95%, 100% EtOH, each others 5min 10. Prewarmed PCR plate form : 55, 5min

8 B. FOR PRIMER 1. primer reaction mixture 준비 Primer Primer 용량 Primer : pmol 10x datp : 0.2mM 10x dctp : 0.2mM 10x dgtp : 0.2mM 10x dttp : 0.02mM 50x Biontin-16-dUTP : 0.02mM 10x Buffer : 50mM Primer chromosome 11-5' (50pmol/ul) chromosome 11-3' (50pmol/ul) 10x datp 10x dctp 10x dgtp 10x dttp 50x Biontin-16-dUTP MgCl 2 : 1.5mM 10x Buffer 5ul Tap.polymerase : 2U MgCl 2 3ul 2ul 2ul 5ul 5ul 5ul 5ul 1ul final volume 50ul Tap.polymerase 0.4ul D.W ul Total 50ul 2. Prewarmed 55 hot block C. IN SITU SYNTHESIS 1. primer on slide coverslip 2. PCR Reaction : 55, 7min 72, 15min 3. 반응후 stop solution(500mm NaCl, 50mM EDTA[pH8.0] at 75 for 5min) 에서반응정지. 4. washing : 4x ssc/ Tween 20(0.1%), at RT, overnight, slowly D. DETECTION 1. Blocking : 3%BSA / 4xssc at 37 for 30min 2. FITC conjugated avidine DN(2.5ul/500ul in 1%BSA) at 37 for 1hr 3. Wash : 4x ssc/ Tween 20(0.1%), 3times, each others 5min at Biotinylated anti-avidine (3ul/250ul in 1%BSA) at 37 for 1hr 5. Wash : 4x ssc/ Tween 20(0.1%), 3times, each others 5min at FITC conjugated avidine DN(2.5ul/500ul in 1%BSA) at 37 for 1hr 7. Wash : 4x ssc/ Tween 20(0.1%), 3times, each others 5min at counterstain : DABCO 1ml Propidium iodide 1.25ul( 조절가능 ) PPD 5ul

9 9. mount 10. confocal laser scanning microscope MULTICOLOR PRINS 1. Reaction mixture : digoxigenin-11-dutp 2. Denaturation of chromosome DNA 3. Probe annealing and chain elongation 4. Termination of labeling 5. Dehydration in cold ethanol (70%, 90%, 100%) x 3times, and airdry 6. New reaction mixture : biotin-16-dutp 7. Preheat the reaction mixture and the slide, 1min, Probe annealing and chain elongation 9. Termination of labeling 1min, Washing 11. Detection III. 참고문헌 (FISH, PRINS) 1. Kearney L. and Buckle VJ. New methods in cytogenetics. Current Opinion in Genet and Dev. 1994, 4: Koch J, Hind Kjaer J, Kolvraa S and Bolund L. Construction of a panel of chromosome-specific oligonucleotide probes(prins-primers) useful for the identification of individual human chromosomes in situ. Cytogenet Cell Genet. 1995, 71: Koch J, Kolvraa S, Peetersen K, Gregersen N, Boland L. Oligonucleotide priming methods for chromosome specific labeling of alpha setellite DNA in situ. Chromosome. 1989, 98: Schrock E, Manoir S, Veldman T et.al. Multicolor Spectral karyotyping of human chromosome. Science. 1996, 273: Speicher MR, Ballard SG, Ward DC. Karyotyping human chromosome by combinatorial multi-fluor FIS. Nature gen. 1996, 12: Human chromosomes : Principles and techniques 2nd ed. 1995, pp Mc Graw Hill.

10 본글의저작권은 " 생물학전문연구정보센터 Biowave" 에있습니다. 일부내용인용시 " 생물학전문연구정보센터 BioWave ( 년 8 월호 " 로정보출처를밝혀야합니다. 전체내용에대한인용시생물학전문연구정보센터의사전허락 (mail: biowave@bric.postech.ac.kr Tel: ~8) 을받으신후전재가가능합니다. ( 단. 원저작자의경우는정보출처만밝히시면됩니다.)

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