аЯрЁБс>ўџ <>ўџџџ;џџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџьЅСq ПЩbjbjt+t+ 1BAAГџџџџџџ]8\V№РРРРРРРРѕ ї ї ї F= Кї КБ $Fє:~е РРРРРе <РРР<<<Р|РРѕ Рѕ <H<„ е ѕ Р”,€aШkюЭТ<э RNA FISH (Sarah Duthie) DAY 1 FIXING CELLS* *all solutions must be made with RNAse-free water and all glass dishes must be baked. Wear gloves! Grow cells on slides in square petri dishes (eg Superfrost Plus autoclaved glass slides - treated with 0.1% gelatin if cells have problems adhering- or plastic LabTek chamber slides from Nunc) Rinse slides in 1XPBS at RT in a coplin jar Fix for 20-30 min in 4% formaldehyde/5% acetic acid/0.9% NaCl at 40C ReagentFinal conc37% formaldehyde10.8 ml16.2 ml4%100% acetic acid5 ml7.5 ml5%NaCl0.9 g1.35 g0.9%H2OUp to 100 mlUp to 150 ml Wash slides 3x in PBS and store in 70% ethanol at 40C Before hybridisation dehydrate cells for 2 min each in 70%, 90% and 100% EtOH and dry in a vacuum dessicator Wash for 5 min in 100% xylene (in fume hood) to remove residual lipids Rehydrate with 100%, 90% and 70% EtOH and then place in PBS Treat with 0.01% pepsin in 0.01M HCl for 5 min at 370C For 300 ml H2OFor 50 ml H2OConc. HCl258 m№l43 m№l10% pepsin stock300 m№l50 m№l Wash for 5 min in PBS on shaker Post-fix in 1% formaldehyde for 10 min in fume hood 8.1 ml of 37% stock in 300 ml H2O or 1.35ml of 37% stock per 50 ml) Wash for 5 min in PBS on shaker Dehydrate in EtOH series as before and dry in dessicator IN-SITU HYBRIDISATION Hybridisation mix (10 m№l/slide): Reagent200m№lFinal conc.Formamide100 m№l50%20xSSC20 m№l2x0.5M EDTA4 m№l10 mM50% dextran sulphate20 m№l5%1M NaH2PO4, pH 7.45 m№l25 mM5M NaCl12 m№l0.3 MH2O39 m№l Probe preparation (per slide): 50 ng DNA probe 5 m№g salmon sperm DNA (10mg/ml, Boehringer MB grade) 20 m№g yeast or E.coli tRNA Add 2 vol of 100% ethanol and dry in speed vac completely Resuspend in 10 m№l/slide hyb mix and add 1m№l RNAguard Leave to dissolve at 37АC for 30 min at least Denature probe before use at 80АC for 10 min Keep probe on ice until needed Warm slides and coverslips to 37АC on hotplate Add 10 m№l of probe per 22x22 mm coverslip Seal the coverslips with cow-gam, put slides in a sandwich box, cover with foil and incubate in a water bath o/n at 37АC DAY 2 WASHES 2xSSC/50% formamide* 3 x 3 min each at RT Cover slips should fall off in 1st wash - be careful not to scratch slide *eg. 50ml of 2xSSC and 50 ml of 100% formamide 2xSSC/50% formamide 1 x 3 min at 37АC 2xSSC 1 x 3 min at RT Transfer to a jar with 4xSSC/0.1%Tween DETECTION Make antibody dilutions for detection of biotin and digoxygenin in blocking buffer (4xSSC, 5% skimmed milk); if detect both at the same time make sure that antibodies are not cross-reacting Mix well and spin down at 14000rpm for 10-15 min at RT; keep ABs at RT in the dark before use Shake off excess fluid from the slide but never allow to dry; Incubate slide in 40 m№l blocking buffer (also spun down) under a 24x40 mm coverslip, 30 min 37АC, humid chamber Shake off coverslip and incubate in 40 m№l 1st antibody 30 min 37АC, humid chamber Wash 3 x 4xSSC/0.1%TWEEN with agitation at 37АC Repeat the procedure with all following antibody layers After last antibody, wash 3 x 4xSSC/0.1%TWEEN with agitation at 37АC Wash 1xPBS mount in 10-15 m№l Vectashield (antifade mounting medium, Vector labs, H-1000), containing 0.1 m№g/ml DAPI (from a 1000 x stock of DAPI already in vectashield at 0.1 mg/ml) and cover with a 22x22 mm coverslip (squash out excess+bubbles). Eg. Antibody layers for simultaneous biotin and digoxygenin detection ProbeLayer 1Layer 2Layer 3Layer 4Layer 5BiotinBB-AV-TRBAAAV-TRDigoxygeninAD-FITCFITC-AS-- Dilutions: AD-FITC (anti-dig FITC, made in sheep) 1:15 FITC-AS (FITC-anti-sheep, made in rabbit) 1:100 AV-TR (avidin-texas red) 1:500 BAA (biotinilated anti-avidin, made in goat) 1:100  ,ŽМQСТЦ;<ZŽЙКНЪЫий >@JLR B D Œ H t v Ђ Є И а в №  їяхсйвЫФЫМЫФДФЫМЫМЫФДФДФ­Ф­Ф­Ф­ФЫЅ›ЅЫ”хŠх‚z‚Ф­5CJOJQJ5CJOJQJ5>*CJOJQJ CJOJQJ6CJH*OJQJ6CJOJQJ CJOJQJCJH*OJQJCJH*OJQJ CJOJQJ CJOJQJ CJOJQJ6CJOJQJ5CJ5>*CJOJQJ>*CJOJQJ5CJOJQJ0 ,QR~ХЦЮЯалмэѕ§§јѓяыыфыфыфыппппУ”ппппУ„$$–lжж SН яЫˆ!„;ў$ & F„;ў„;ў„;ў$„;ў$„;ў ,QR~ХЦЮЯалмэѕ§!"'-49:>KXY§њїєюыуриеаЫЦСНИГЎЉЅ ›–‘ˆƒ~yupkfa  Ц§џџ  г§џџ  р§џџ  ф§џџх§џџ  ъ§џџ  ё§џџ  ї§џџ  ќ§џџ§§џџ  ўџџ  ўџџ   ўџџ  ўџџўџџ  !ўџџ  )ўџџ  1ўџџ  BўџџCўџџ  Nўџџ  Oўџџ  Pўџџ  XўџџYўџџŸўџџ   ўџџЬўџџ  Эўџџ Žџџџ џџџђџџџ$!"'-49:>KXYZ[‘’џGH„…њњњњо`њњњњо€њњњњокгкгкгкгк & F„;ў„;ў$$–lжж SН яЫˆ!„;ў$YZ[‘’џGH„…МНОЭлмцъ6DPRV–˜  Œ Ž Ю а B D H t v И К Ъ ж ю ћј№эхткзЯЬФСМЗВЎЉЇЇЄЂЄЄЄЂœ–Š‡„~xr  Иџџџ   Оџџџ   Цџџџ Чџџџ            BќџџCќџџ  Qќџџ  `ќџџ  aќџџbќџџ™ќџџ  šќџџжќџџ  зќџџ§џџ  §џџŒ§џџ  §џџУ§џџ  Ф§џџХ§џџ,…МНОЭлмц6DPRV–˜ јєяяяЖ`яяя›|яяя›єјєј$$–lжж SН Y8$$–lжжFSН Yџџџџџџџџџџџџ„;ў$„;ў & F„;ў  Œ Ž Ю а B D H t v И К Ъ ж ю №     * 6 < ћѕћюћюћћьћћћчттЧXчттЧDчтт$$–lжж Sf&Y„фў$„;ў$ & F„;ў„;ў„S„;ўю №     * 6 < > R \ h j ”   І Ј Ю и ф ц і     $ & ( * , j l Ž ј . 0 Є І њєюшунзбЬЦРКЕЏЉЃž˜’Œ‡{wtwwwtrlf          (џџџ   0џџџ  1џџџ   7џџџ   <џџџ   Oџџџ  Pџџџ   Sџџџ   Yџџџ   nџџџ  oџџџ   uџџџ   zџџџ   „џџџ  …џџџ   ˆџџџ   Žџџџ   •џџџ  –џџџ   šџџџ   Ёџџџ   Ћџџџ  Ќџџџ ( ( * 0 2 4 6 < > V X h j ’ ” š œ ž   І Ј Д Ж К М в д ф ц ќ ў        " ( * , R f h j ’ ” ў Ц Ш њ ќ ‚„agno™ф:<ABљяљшљяљяљшљяљяљшљяљяљрљрљшљяљшљяљрљяљшљяжЯжЧжПЯИЯИЯИЯИЯИЯЖЯПЯЎЯПЯПЯ6CJOJQJ5 CJOJQJ5CJOJQJ>*CJOJQJ CJOJQJ5>*CJOJQJCJH*OJQJ CJOJQJ5>*CJOJQJ CJOJQJD< > R \ h j ”   І Ј Ю и ф ц і     $ & ( * , j фXпккф|пккф|пккфTпккф0пккфжжж„;ў„фў$„;ў$$$–lжж Sf&Yj l Ž ј . 0 Є І rtавrtШЪ^_agno™ћѓѓѓћьћьћьћьћьћьћьћьћћщчћь$ & F„;ў„;ў„Х„Х„;ўrtавrtШЪ^_agno™уф;<RSz{|†‡ŠŒHJЦШЈЊNPАВ"$ЎАШЪQRS™š њєюштмйжгЫШХТПЗДЌЉЁž›•’йŒ  ѕџџџ ъўџџыўџџьўџџџџџ  џџџ*џџџ  +џџџQџџџ  Rџџџ‚џџџƒџџџЭџџџїџџџ  јџџџ                  7™уф;<RSz{|†‡ŠŒHJЦШЈЊNPАВ"ѕѕяѕшѕшфшффтфшфшфшфшфшфшфш„;ў & F„;ў„;ў„ќ „;ў„Њ ЦЊBRj{|†Шђєјњшъ†ˆSV™ ШЩї№ї№ь№фмф№е№Э№е№е№Ы№ф№>*CJH*OJQJ CJOJQJ>*CJOJQJ>*CJOJQJ5>* CJOJQJ5CJOJQJ"$ЎАШЪQRS™š ЈАИРШћєћєћєћћђћэццццц$„gџ$„;ў$ & F„;ў„;ў ЈАИРШЩагелпхцђѓћ Bs•ШЩ§§§§§ћ§§§§§§ћ§§§§§§ћј ШЩагелпхЅt ™™™™™$„gџ$„;ў$Y$$–lжжˆ”џ=ц 8сŠ!џџџџџџџџџџџџџџџџџџџџхцђѓћЅˆ ™™™™™$„gџ$„;ў$Y$$–lжжˆ”џ=ц 8сŠ!џџџџ Bs•ШЩЅЁЁЁЁЁЁŸ„;ўY$$–lжжˆ”џ=ц 8сŠ!џџџџ/ 0PАh. АДA!АЅ"АЅ# $ %ААсАс с [(@ёџ(NormalCJmH 8@8 Heading 1$@& 6OJQJ@@@ Heading 2 $„;ў@&6CJOJQJB@B Heading 3 $„;ў@&5>*CJOJQJB@B Heading 4 $„;ў@&6>*CJOJQJB@B Heading 5 $„;ў@&56CJOJQJ<A@ђџЁ<Default Paragraph Font2>@ђ2Title $„;ў 5OJQJГBџџџџ BЩ… < j ™"ШхЩ Yю  ЩЦZ  $%FFўўIJPPБ о œВВЕџџATSGMMacintosh HD:My documents:PROTOCOLS:Cytogenetics:FISH methods:RNA FISH_XI.docATSGPMacintosh HD:My documents:PROTOCOLS:Cytogenetics:FISH methods:RNA FISH_Sarah.docATSGPMacintosh HD:My documents:PROTOCOLS:Cytogenetics:FISH methods:RNA FISH_Sarah.docATSGPMacintosh HD:My documents:PROTOCOLS:Cytogenetics:FISH methods:RNA FISH_Sarah.docATSGPMacintosh HD:My documents:PROTOCOLS:Cytogenetics:FISH methods:RNA FISH_Sarah.docATSGPMacintosh HD:My documents:PROTOCOLS:Cytogenetics:FISH methods:RNA FISH_Sarah.docATSGPMacintosh HD:My documents:PROTOCOLS:Cytogenetics:FISH methods:RNA FISH_Sarah.docATSGPMacintosh HD:My documents:PROTOCOLS:Cytogenetics:FISH methods:RNA FISH_Sarah.docComputing Officer=HARD DISK:Useful bits and pieces:Protocols:RNA FISH_Sarah.docdonaldC:\WINDOWS\Desktop\FISH1.docџџџџџџџџџ„h„˜ўЦho(-џџџџџџџ@EPSON Stylus COLOR 900 (Copy 2)LPT1:EPIJNL20EPSON Stylus COLOR 900EPSON Stylus COLOR 900 (Copy 2)'”&‹€ š 4hh DLLName16=EPIGUE3V.DLLDLLName32=EPIDA230.DLLEPSON Stylus COLOR 900hh L L dhh  q***Ц  q***Ц2џ4š EPSON Stylus COLOR 900 (Copy 2)'”&‹€ š 4hh DLLName16=EPIGUE3V.DLLDLLName32=EPIDA230.DLLEPSON Stylus COLOR 900hh L L dhh  q***Ц  q***Ц2џ4š €ѓuE|ъъъъъъ ъ ъ ъ Г`@`` ` `` @````0@GTimes New Roman5€Symbol3& Arial9New York"€Х­3r†­3r†s*ff  ƒь€+„ЅРДД€0dсџџRNA FISH PROTOCOLPowerMacintosh Userdonaldўџр…ŸђљOhЋ‘+'Гй0„˜ДРмшќ  4 @ L Xdlt|фRNA FISH PROTOCOLdNA PowerMacintosh Userowe Normal.dotodonaldd2naMicrosoft Word 8.0@@Z:v Т@>SюЭТ@>SюЭТ  ўџеЭеœ.“—+,љЎDеЭеœ.“—+,љЎ<ј hp|„Œ” œЄЌД М кфMRCс1 RNA FISH PROTOCOL Title˜ 6> _PID_GUIDфAN{8D1EB701-DC5C-11D5-AA5B-000393102C20}  !ўџџџ#$%&'()*ўџџџ,-./012ўџџџ456789:ўџџџ§џџџ=ўџџџўџџџўџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџRoot Entryџџџџџџџџ РF IЅkюЭТ аkюЭТ?€1Tableџџџџџџџџџџџџ"ИWordDocumentџџџџџџџџ1BSummaryInformation(џџџџ+DocumentSummaryInformation8џџџџџџџџџџџџ3CompObjџџџџjObjectPoolЌЪ@Ќ