1.Foreachprobe(controlandexperimental),setupaseparate100-mlPCRina0.5-mlsteriletube,astabulatedbelow.Either.cDNAinsertedinplasmidsorgenomicDNAcanbeusedastemplatesforthePCR(seeREAGENTSETUPfordetailsonprimerdesign).每個探針(實驗組和對照組),在0.5-ml無菌管設立一個獨立的100毫升PCR,正如下面的表。另外?；パa脫氧核糖核酸插入到基因組DNA質(zhì)體或可用作模板PCR(見試劑設置有關底漆設計)。**注意關鍵：1.很多版本的實驗反義核酸探針可以作為一種控制背景染色(見試劑設置)。然而,我們相信最好的方法來演示特異性是獲取相同的空間限制表達模式使用不同的非重疊探測器相同的基因。2.小心不要污染pcr.使用無菌試管和過濾器的技巧和戴手套3.另外,PCR擴增,cDNAs質(zhì)粒中可以使用約束線性化酶,獨特的站點位于5￠(反義核酸探針)或3￠(對感官探測)來插入。凈化的線性DNA可以通過苯酚/氯仿萃取乙醇沉淀緊隨其后。2|RunthePCRusingtheconditionstabulatedbelow.使用下面列出的條件運行PCR**暫停點：把擴增好的pcr產(chǎn)品放4℃降溫和在-20℃貯藏幾個星期。3|Addthe100-mlPCRtoaMicroconYM-50columnandadd400mlofsterilewater.Centrifugefor15–20minat1,000gatroomtemperature.加入100毫升PCR到MicroconYM-50列并加入400毫升的無菌水。在室溫下1000g離心15-20分鐘。**注意關鍵：膜應該是干的。如果沒有再離心4|PlacetheMicroconcolumnintoanewmicrofugetube(providedinthekit),add20mlofsterilewater,vortexbrieflyandthenturntheMicroconcolumnupsidedown.Spinfor1minat1,000gatroomtemperaturetorecovertheDNA.把小層析柱放在一個新的離心管(在這個工具包中提供),增加20毫升的無菌水、短暫離心,然后顛倒層析柱。自旋1分鐘1000g在室溫下恢復了DNA**注意關鍵：離心的步驟應該快速。離心機1分鐘只是為了避免樣本太干。5|Checkthequality,quantityandsizeofthePCRamplificationproductbyloading1/20ofthepreparationona1%(wt/vol)agarosegelin1TBEbuffer.DNAshouldappearasabandandnotasasmear.The1/20ofthepreparationshouldcontainatleast40ngofDNA.通過裝載1/20的稀釋液在1*的TBEbuffer緩沖液中的1%(wt/vol)的瓊脂糖凝膠檢查PCR的擴增產(chǎn)物的質(zhì)量數(shù)量和大小。DNA產(chǎn)物應該以一個條帶而不是以一塊污點表現(xiàn)出來。這份1/20的稀釋液至少含有40ng的DNA。6|Foreachprobe,addthecomponentstabulatedbelowtoamicrofugetube.Mixandincubatefor2hat371C.按下面表格給每一份在離心管中的談增加成分，混合均勻后放在37℃下保存2h.7|Add2mlofRNase-freeDNaseIand18mlofsterilewater.Mixandincubatefor30minat371C.添加2毫升的RNase-freeDNaseI和18毫升的無菌水。在37℃下混合和孵化30分鐘。8|Stopthereactionbyadding1ulofsterile0.5MEDTAand9ulofsterilewater.停止反應，通過添加1微升的無菌0.5MEDTA和9微升無菌水。9|PlaceaSigmaspinpost-reactionpurificationcolumnontopofamicrofugetube.Centrifugefor15sat750g.放一個Sigmaspin反應純化柱到小離心管，750g離心15s。10|Breakthebaseofthecolumnanddiscardthelid.Spinfor2minat750g.破壞柱子的基柱，丟棄蓋子，750g離心旋轉(zhuǎn)2min11|Placethecolumnonanewmicrofugetube.AddtheRNAtemplateontopoftheresin.Centrifugefor4minat750g.Discardthecolumn.把柱子放在一個新的離心管上，在樹脂上加上RNA模板。750g離心4min，丟掉柱子。12|Add1mlofsterileEDTA0.5Mand9mlofRNAlatertothesample;thisprotectstheRNAfromdegradation.加1ml無菌的EDTA0.5M和9mlRNAlater到體系里；這樣可以防止RNA降解**注意：由地高辛標記的反義（或正義）的RNA能在-20℃環(huán)境下保存數(shù)月。這個探針合成提供了足夠的反義(或正義)RNA執(zhí)行至少40個左右的不同原雜反應。13|Visualize1/20ofthesynthesizedRNAon1%(wt/vol)agarosegelin1TBEbufferafter30minofelectrophoresisat230V.Agoodprobeshouldappearasoneortwodiscretebandsonthegel,notasasmear—whichwouldindicatedegradation.設想1/20的合成RNA稀釋液在1*的TBEbuffer緩沖液中的1%(wt/vol)的瓊脂糖凝膠在230V下經(jīng)過30min的電泳后，好的探針應該以一到兩條條帶在膠上，而不是一塊污點，用來顯示降解。14|Setuppairsofmaleandfemalefishinbreedingtanksthenightbeforeeggsaretobecollected.Separatethemaleandfemalewithadiagonalplasticdivider.在魚卵收集前的晚上建立一個飼養(yǎng)雄魚和雌魚的缸，用一個塑料對角分隔器將雄魚與雌魚分開飼養(yǎng)。15|Thenextmorning,whenthelightgoeson,placetheupperpartofthebreedingtankinacleanlowerpartfilledwithfreshwater.Removethedividerofthebreedingtankandletthefishmatefor10–20min.Theeggslaidwillsinktothebottomofthebreedingtank.第二天天亮后，把飼養(yǎng)缸上半部分移到一個新鮮的淡水體里。移動分離器，讓魚交配10–20min。魚卵就會沉到水底。**注意：成人斑馬魚在野外交配時通常在廣下。在養(yǎng)魚設施中,絕大多數(shù)的魚產(chǎn)卵5min到1h后把燈打開。可以在上午晚些時候可以獲得額外的蛋,但它們通常質(zhì)量低劣。16|CollecttheeggsfromthebottomtankbypouringthemintoPetridishes.從水缸里收集沉在水底的魚卵，放至培養(yǎng)皿里。**注意：避免一個培養(yǎng)皿中放太多的魚卵17|Cleantheclutchunderastereomicroscopewithadiascopicstand;discarddirtandunfertilizedeggsusingaPasteurcapillarypipette(withalargeopening)andpipettepumpfiller.清理收集魚卵的培養(yǎng)皿，用消毒過的移液器（有大口）吸掉壞的魚卵或未受精的魚卵。18|Placetheembryosina100-mlbeaker,coveredwithaminimalamountofwater(10ml).把有用的胚胎放到一個100-ml大燒杯，燒杯里加合適的最少量水10-ml。19|Pour3mlofpronase(1/100wt/vol,warmedto28.51C)intothebeakerandincubatefor1min.Gentlepronasetreatmentprogressivelysoftensthechorionwithoutdamagingtheembryos.Alternatively,embryoscanbedechorionatedbyhandusingsharpenedforceps,butthisisaslowprocess,whichcanbeusedonlyforasmallquantityofembryos.倒3毫升鏈霉蛋白酶(1/100wt/vol,warmedto28.51C)放入燒杯，孵化1分鐘。溫柔的鏈霉蛋白酶處理逐漸軟化絨毛膜不破壞胚胎?；蛘?用手去卵膜，胚胎能夠使用尖銳的鉗,但這是一個緩慢的過程,可以僅用少量的胚胎。20|Afterthe1-minincubation,fillthebeakerwithfishwater.Lettheembryossinktothebottomofthebeaker,thenslowlypourthewaterout.經(jīng)過1-min的潛伏期，用魚水將燒杯加滿，讓胚胎沉到燒杯的底部，緩慢倒出水。21|Gentlyrinsetheeggsthreetimeswithfishwater.Somechorionswillalreadyhavebeenremovedbythisstage.輕輕地用魚水沖洗魚卵三次，一些絨毛膜就會在這個階段被洗脫。22|PlacetheembryosinlargePetridishes(maximum50embryosfora94-mmPetridishand600embryosfora145-mmPetridish)coatedwith2%agarose.用2%瓊脂糖覆蓋后，把胚胎放到大的培養(yǎng)皿里面。23|Lettheembryosdevelopat28.51Cuntilmostofthechorionshavebeenremoved.CollectthedechorionatedembryosbypouringtheminanewPetridish.等到大部分的絨毛膜脫落時，把胚胎放到28.5℃進一步發(fā)育。收集無絨毛膜的胚胎倒到一個新的培養(yǎng)皿中。**注意：鏈蛋白酶使絨毛膜變得柔和，所以，過了這個階段，將近一般的絨毛膜再沖洗后會脫落。用一個小口的移液器小心的移取絨毛膜，可以去除幾乎所有絨毛膜。胚胎易碎，小心操作。24|Continuetoincubatetheembryosat28.51CinPetridishescontainingfishwateruntilthedesireddevelopmentalstageisreached.Ifpost-gastrulationstagesaretobeexamined,theformationofmelaninpigmentneedstobeprevented.Thiscanbeachievedbyreplacingregularfishwaterwith0.0045%PTUsolutionpreparedin1Danieaumedium13attheendofgastrulation.Forlarvaeandembryosolderthan24hourspost-fertilization(hpf),changethismediumonceaday.繼續(xù)在28.5℃的含魚水的培養(yǎng)皿中培養(yǎng)胚胎，直到達到預期的成長階段。如果柱原腸胚形成階段是要審查,那么黑色素的形成需要避免。這樣的期望在用1*Danieaumedium的0.0045%PTU溶液定時的替換魚水可以在原腸胚后期實現(xiàn)。對于超過1hpf的幼體和胚胎，每天換一次媒介Danieaumedium25|Fixdechorionatedembryosoftheappropriatedevelopmentalstage(s)in4%(wt/vol)paraformaldehydein1PBSovernightat41CintoPetridishes.在適當?shù)陌l(fā)育階段混合無絨毛膜的胚胎，添加4%(wt/vol)多聚甲醛混于1*PBS溶液后，放4℃的培養(yǎng)皿中一晚上。**注意：多聚甲醛溶液不應超過兩天，最好現(xiàn)配先用。26|Thenextday,dehydratetheembryosin100%methanolfor15minatroomtemperature.IftheembryoswerenottreatedwithPTUatStep24,removepigmentationwithH2O2asdescribedinBox1第二天，用100%甲醇溶液把胚胎在室溫下脫水15min。如果胚胎在Step24時不與PTU反應，則像Box1描述的那樣用H2O2溶液清除染色液。27|Placetheeggsat-20℃in100%methanolforatleast2hbeforeuse.用之前把魚卵放在100%甲醇溶液-20℃環(huán)境下保持至少2h.（胚胎可以在-20℃下保存幾個月）28|Transferdehydratedembryosofthesamedevelopmentalstagetobasketsmadeofstainlesssteelmesh(largebasketfortreatmentof500upto1,000embryos)ormadeofnylonmesh(smallbasketfortreatmentofupto50embryos).Smallbasketsfilledwithembryosareplacedinto24-wellplatesandlargebasketsareplacedintosix-wellplates(seeFigs.3aand5).Thisandthefollowingstepsareallperformedatroomtemperature.在相同的發(fā)育階段轉(zhuǎn)移脫水胚胎到不銹鋼網(wǎng)籃子(大籃子可以處理500到1000胚胎)或尼龍網(wǎng)小籃子(小籃子可處理50胚胎)。放滿了胚胎被放置到24孔板或大籃子被放置到六個孔板(seeFigs.3aand5)。這個步驟和下面的步驟都是在室溫下進行。29|Rehydrateembryosbymovingthebasketfromonewelltothenextwelloftheplateintosuccessivedilutionsofmethanolin1*PBS:5minin75%(vol/vol)methanol;5minin50%(vol/vol)methanol;and5minin25%(vol/vol)methanol(seeFig.5).通過連續(xù)的1*PBS稀釋甲醇液給無水胚胎充水，5minin75%(vol/vol)甲醇;5minin50%(vol/vol)甲醇;and5minin25%(vol/vol)甲醇(seeFig.5).30|Washfourtimes,5minperwash,in100%PBT.用100%PBT洗5次，每次15分鐘。31|PermeabilizetheembryosbydigestionwithproteinaseK(10mgml1)atroomtemperatureforthetimeindicatedinthetablebelow.按下表操作，用蛋白酶K(10ug/ml)在室溫下使胚胎消化。**注意：這一步消化胚胎和允許RNA探針的滲入，對于蛋白酶K的反應用好這個表很重要，一定要在之前小心的判定胚胎的發(fā)育階段。正在消化的胚胎是不會允許探針滲入的，而消化后會改變胚胎的形態(tài)。32|StoptheproteinaseKdigestionbyincubatingtheembryosfor20minin4%(wt/vol)paraformaldehydein1*PBS.把胚胎放在混于1*PBS中的4%(wt/vol)多聚甲醛溶液中孵育20min，以停止胚胎的消化。33|Washfourtimes,5minperwash,in1*PBTtoremoveresidualparaformaldehyde.用1*PBT溶液洗脫殘留的多聚甲醛，多次洗脫，每次5min.34|Transfertheembryosfromtheirbasketsto1.5-mlsterileEppendorftubes(upto50embryospertube).Atthispoint,embryosofdifferentdevelopmentalstagescanbepooledforagivenprobeuntiltheendofprotocol.把胚胎轉(zhuǎn)移到1.5ml離心管中（每管至少50個），這時，不同發(fā)育階段的胚胎都會滲入探針直到這個步驟結(jié)束。35|Prehybridizetheembryoswith700mlHMfor2–5hina701Cwaterbath.。預雜交：加700mlHM到Prehybridize胚胎70℃水浴2–5h。（Prehybridize胚胎可以在HM中20℃保存至少幾周。）36|DiscardHMandreplacewith200mlofHMcontaining30–50ngofantisenseDIG-labeledRNAprobe(fromStep12).Hybridizeovernightat701C.用包含30–50ng的反義地高辛標記的RNA探針的200mlHM替換原管中的HM，70℃雜交一晚上。**注意：1）不要用過度數(shù)量的探針，這樣會增加背景染色。2）HMbuffer中的高雜交溫度和濃的甲酰胺，確保了高雜交率和避免了交叉雜交的產(chǎn)生。37|TransfertheembryosinHM-containingprobetosmallbasketsplacedonastyrofoamfloat(16cm*9cm*1.5cmwithspacefor50smallbaskets)inaplasticbox(21cm*10cm*7cm)filledwith200mlofHM(withoutRNase-freetRNAandheparin)warmedat701C,usingapipetteranda1-mlpipettetip(cutwithascalpeltogeta2-mm-wideopening).轉(zhuǎn)移包含探針的HM中的胚胎倒小離心管里，放到泡沫浮板（16cm*9cm*1.5cmwithspacefor50smallbaskets）上在一個塑料箱(21cm*10cm*7cm)裝上200mlofHM(不含RNase-freetRNAandheparin)在70℃加熱，用一個移液器和一個1ml移液管（用小刀切開一個2mm的開口）。**注意：讓胚胎呆在塑料箱中的浮板直到第47步。(Step47)(seeFig.3c).所有溶液在用之前都放在70℃下加熱。38|GraduallychangetheHMto2*SSCthroughaseriesof10min,701CwashesinHMdilutedwith2*SSC.Washonceineachofthefollowing:75%HM,50%HM,25%HMand100%2*SSC.Performwashesina701Cwaterbathwithgentleagitation.通過一系列10min，70℃水洗用2*SSC把HM沖淡的方法逐步的把HM換成2*SSC。**注意：HM適用于水洗液中不含有tRNA和肝素。39|Washtwice,for30minperwash,in0.2*SSCat701C.用0.2*SSC70℃水洗兩次，每次30min。**注意：這種高頻率的水洗避免了非特異性的探針雜交。40|Progressivelyreplace0.2*SSCwithPBTthroughaseriesof10-minwashesin0.2*SSCdilutedwith1*PBT.Washonceatroomtemperatureonahorizontalshaker(40r.p.m.)in200mlofthefollowingsolutions:75%0.2SSC,50%0.2SSC,25%0.2*SSCand1*PBT.通過一系列的用1*PBT稀釋的0.2*SSC清洗十分鐘的洗脫，逐步的用PBT溶液替換0.2*SSC。每次洗的時候都在室溫下的水平搖床(40r.p.m.)進行，并且加上一下溶液：75%0.2SSC,50%0.2SSC,25%0.2*SSCand1*PBT.41|Incubatetheembryosfor3–4hatroomtemperatureinblockingbuffer.在室溫下在整批緩沖液中孵育胚胎3-4h.（這一步，飽和烴非特異的結(jié)合在抗體上。）42|Incubatein200mlofanti-DIGantibodysolutiondilutedat1/10,000withblockingbufferovernightat4℃withgentleagitation(40rpm.onahorizontalorbitalshaker).Washesandstaining
TIMING4–11h(day3)在200ml的blockingbuffer稀釋的抗地高辛抗體1/10,000稀釋溶液里4℃孵育一晚上，而且在水平搖床上（40rpm），清洗染色（定時4–11h）。43|DiscardtheantibodysolutionandwashtheembryosbrieflyinPBT.倒掉抗體溶液，在PBT溶液中短暫的清洗胚胎。44|Washsixtimes,15minperwash,inPBTatroomtemperaturewithgentleagitation(40r.p.m.onhorizontalorbitalshaker).在水平搖床（40rpm）上，用PBT溶液室溫下清洗六次，每次十五分鐘。45|Brieflydrytheembryosbyplacingthestyrofoamfloatcarryingthebasketsonasheetofabsorbentpaper.短暫的干燥胚胎，用把泡沫浮板帶離心管到一張吸水紙上。**注意：這一個重要步驟是避免在堿性的Trisbuffer里面形成沉淀物導致粘連胚胎。46|Incubatetheembryosatroomtemperaturethreetimes,5minperwash,inalkalineTrisbufferwithgentleagitation(40r.p.m.onhorizontalorbitalshaker).室溫下堿性Trisbuffer中孵育胚胎三次，每次五分鐘。并且在水平搖床（40rpm）上。47|Transfertheembryosfromthebasketsto1.5-mlmicrofugetubes.轉(zhuǎn)移胚胎至1.5ml離心管中。48|RemovethealkalineTrisbufferandreplacewith0.7-mlstainingsolutionpreparedfreshandkeptinthedark.用0.7ml新鮮配制的染色液替換堿性Trisbuffer，并且放置在避光環(huán)境中。**注意：染色液應該是淡黃色，若是粉紅色，則配制一份預防背景染色液。49|Transfertheembryostospotplates(seeFig.6).Monitorthecolorreactionperiodicallyunderadissectingscope,litfromabove.Keeptheembryosinthedarkbetweenchecks.?50|Whenthedesiredstainingintensityisreached(reactiontimeintherangeof15minforhighlyexpressedgenes,1–1.5hforthemajorityofgenestestedandupto8hforweaklyexpressedgenes),stopthereactionbytransferringtheembryosto1.5-mlEppendorftubes,discardingasmuchofthestainingsolutionaspossibleandfillingthetubewith1-mlstopsolution.Washthreetimesfor15minatroomtempera-tureonatesttuberockerwithgentleagitation.’PAUSEPOINTStainedembryoscanbestoredinthedarkinstopsolutionat41Cforseveralmonths.Mountingembryos
51|Ifstudyingearlydevelopmentalstages,incubatetheembryosfor5minin1PBS,pH3.0.Otherwise,proceedimmediatelytoStep52.mCRITICALSTEPBythisstageintheP