斑馬魚胚胎取樣

1、JNKs在斑馬魚胚胎發(fā)育過程中的分化表達(dá)及功能研究_鄒立軍在室溫25e下,斑馬魚發(fā)育進(jìn)程:兩細(xì)胞時(shí)期.,1一1.5小時(shí)多細(xì)胞時(shí)期.,2.5一3小時(shí)囊胚期.,6.5一7小時(shí)原腸胚時(shí)期.,8一9小時(shí)神經(jīng)胚時(shí)期.,.,11一11.5小時(shí)視原基時(shí)期(眼原基開始出現(xiàn)).,16.5一17.5小時(shí)腦泡分化時(shí)期(腦部開始形成).,23一23.5小時(shí)肌肉效應(yīng)時(shí)期(胚胎開始有肌肉運(yùn)動(dòng)).,28一28.5小時(shí)心跳時(shí)期.,32一33小時(shí)眼色素時(shí)期.,36一36.5小時(shí)體色素時(shí)期.,40一42小時(shí)出膜時(shí)期(成體).,48一50小時(shí)原位雜交：取不同時(shí)期受精卵十枚，置于4%多聚甲醛（含DEPC）固定后去卵膜。（4度過夜或者

2、室溫三小時(shí)）Wash with 50% MeOH/PBST (PBST= PBS + 0.5-0.8% TX-100)Wash 2x with 100% MeOH Store at -20°C in MEOH at least overnight (up to 6 months)DAY1. Rehydration of embryos 8. Wash for 5min each in MEOH: PBST. 3:1, 1:1, 1:3 9. Wash 4X, 5min each in 100% PBST 10. Incubate in proteinase K(dilute 1mg/

3、l stock 100 fold, 10ug/ml PBST) Younger than “bud” 30min Early somitogenesis 1minute Late somitogenesis 2minutes 24hpf 6minutes 36/48hpf 12minutes 11. Wash once(quick) in PBST to get rid of the proteinase K(optional) 12. Refix for 20min in 4% p-formaldehyde at RT 13. Rinse 5X 5min in PBST. Hybridiza

4、tion 14. Prehybridize embryos in hybe+ buffer at 70 for 30min-3hours 15. Replace prehybe with hybe+ buffer containing the probes of choice (300ng probes/ 200ul hybe+buffer) 16. Incubate at 70 DAY2 17. Remove hybe/probes mixture and store at 20 18. Washs: Wash：50% formamide; 2xSSC; 0.1% Tween20 15min

5、,2X Wash：2xSSC; 0.1% Tween20 15min,1X Wash：0.2xSSC; 0.1% Tween20 30min,2X 19. Replace wash buffer with MABT for 3times, 5min each, at RT. 20. Block embryos in MABT:blocking:FBS=7:2:1 buffer at RT for 1hour at least. Antibody incubation 21. Incubate embryos with 500ul of antibody solution(1:3000 dilu

6、tion) o/n, rocking on a platform, at 4DAY3. Dig staining 22. Wash quickly in MABT 23. Wash 5X in MABT, shaking at RT, 1hour each times 24. Wash 3times, 5min in staining buffer (fresh made) 25. Add BCIP/NBT in staining buffer ,store at 4overnight.DAY4 26. Stop embryos in PBST 27. Wash in PBST : MEOH.

7、1:1, 2X, 5min 28. Wash in MEOH for several times 29. Store embryos in 4% PFA overnight at 4Staining buffer:10ml 1M Tris pH 9.5; 5ml 1M MgCl2; 2ml 5M NaCl; 500ul Tween 20; dH2O up to 10mlBlocking solution:MABT: blocking: FBS=7:2:120X SSC87.65g NaCl61.15g Na3citrate.2H2OpH7.0 up to 500ml10X PBS80g NaCl2g KCl36.3g Na2HPO4.12H2O2.4g KH2PO4100mM Maelic acid150mM NaCl0.1% Tween20Hybe+50% For