斑马鱼胚胎取样.docx

JNKs在斑马鱼胚胎发育过程中的分化表达及功能研究_邹立军

在室温25e下,斑马鱼发育进程:
两细胞时期................................................,,1一1.5小时
多细胞时期................................................,,2.5一3小时
囊胚期......................................................,,6.5一7小时
原肠胚时期................................................,,8一9小时
神经胚时期.............................,,,.....,,,,,11一11.5小时
视原基时期(眼原基开始出现).....................,,16.5一17.5小时
脑泡分化时期(脑部开始形成).....................,,23一23.5小时
肌肉效应时期(胚胎开始有肌肉运动)............,,28一28.5小时
心跳时期...................................................,,32一33小时
眼色素时期................................................,,36一36.5小时
体色素时期................................................,,40一42小时
出膜时期(成体).......................................,,48一50小时
原位杂交：
取不同时期受精卵十枚，置于4%多聚甲醛（含DEPC）固定后去卵膜。（4度过夜或者室温三小时）
Washwith50%MeOH/PBST(PBST=PBS+0.5-0.8%TX-100)
Wash2xwith100%MeOH
Storeat-20°CinMEOHatleastovernight(upto6months)
DAY1
Ⅲ.Rehydrationofembryos
8.Washfor5mineachinMEOH:PBST...3:1,1:1,1:3
9.Wash4X,5mineachin100%PBST
10.IncubateinproteinaseK(dilute1mg/lstock100fold,10ug/mlPBST)
Youngerthan“bud”30min
Earlysomitogenesis1minute
Latesomitogenesis2minutes
24hpf6minutes
36/48hpf12minutes
11.Washonce(quick)inPBSTtogetridoftheproteinaseK(optional)
12.Refixfor20minin4%p-formaldehydeatRT
13.Rinse5X5mininPBST
Ⅳ.Hybridization
14.Prehybridizeembryosinhybe+bufferat70℃for30min-3hours
15.Replaceprehybewithhybe+buffercontainingtheprobesofchoice(300ng
probes/200ulhybe+buffer)
16.Incubateat70℃

DAY2
17.Removehybe/probesmixtureandstoreat20℃
18.Washs:
WashⅠ：50%formamide;2xSSC;0.1%Tween2015min,2X
WashⅡ：2xSSC;0.1%Tween2015min,1X
WashⅢ：0.2xSSC;0.1%Tween2030min,2X
19.ReplacewashbufferwithMABTfor3times,5mineach,atRT.
20.BlockembryosinMABT:blocking:FBS=7:2:1bufferatRTfor1houratleast
Ⅴ.Antibodyincubation
21.Incubateembryoswith500ulofantibodysolution(1:3000dilution)o/n,rockingona
platform,at4℃

DAY3
Ⅵ.Digstaining
22.WashquicklyinMABT
23.Wash5XinMABT,shakingatRT,1houreachtimes
24.Wash3times,5mininstainingbuffer(fres