आर्गेनाइज़र और Xenopus laevis भ्रूण से पशु ध्रुव Explants और एक कोशिका आसंजन परख की सभा के विच्छेदन
Summary
यह वीडियो और भौं चाकू के उपयोग सहित Xenopus laevis भ्रूण, आयोजक जानवर पोल explants की तैयारी के लिए तकनीक का इस्तेमाल एक विशेष विच्छेदन एक भौं का बना उपकरण को दर्शाता है. एक आसंजन परख कोडांतरण के लिए प्रोटोकॉल भी दिया जाता है, जो प्रमुख आसंजन अणुओं की उपस्थिति के लिए जांच सतह आयोजक या जानवर पोल कोशिकाओं है कि समुचित विकास के लिए महत्वपूर्ण हैं पर मौजूद है.
Protocol
तैयारी आसंजन चेम्बर्स (पहले दिन / इंजेक्शन विच्छेदन) आसंजन परख चेम्बर्स यदि आप एक पारंपरिक परिसर माइक्रोस्कोप (चरण ऊपर ऑप्टिकल रेखांकित) है, यह ncecessary है बनाने के लिए एक कम ऊंचाई आसंजन ?…
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 1x MBS-H | Buffer | Modified Barth’s medium: 1L recipe 5.14g NaCl 75mg KCl 0.2g NaHCO3 0.2g MgSO4•7H2O 60mg CaCl2•2H2O 80mg Ca(NO3)2•4H2O 2.38g HEPES, free-acid Adjust the pH to 7.4 with NaOH. Autoclave. | ||
| CMF-MBS | Buffer | This is Ca2+/Mg2+-free version of MBS-H. 500ml recipe : 2.57g NaCl 37.5mg KCl 0.1g NaHCO3 1.19g HEPES, free-acid Adjust the pH to 7.4 with NaOH. Autoclave. | ||
| 1% agarose/1x Barth’s plates | as many as the number of samples | |||
| 1% agarose/CMF-MBS plates | as many as the number of samples | |||
| E-Cadherin/Fc chimera | Reagent | Sigma | E-2278 | Dissolve 0.1mg of solid E-cadherin in 100ul of 1x MBS-H containing 40% glycerol to make 100x master stock (1.0mg/ml). Store it in –20C. Make 1/100 dilution with 1x MBS-H to make 10ug/ml working solution just before you use. You need 200ul per samples of this working solution. |
| Fibronectin, 0.1% solution | Reagent | Sigma | F-1141 | This pre-made solution can be used as 50x stock. Make 1/50 dilution with 1x MBS-H make 20ug/ml working solution just before you use. You need 200ul per samples of this working solution. I do NOT recommend using solid Fibronectin, because it is difficult to dissolve at 0.1% concentration to make the stock solution. Although it is more expensive, this pre-made 0.1% solution is better to obtain consistent results. |
| 4% BSA /MBS-H | Buffer | BSA: molecular biology grade fraction V | ||
| Sigmacoted yellow tips | Tool | |||
| No.1 cover slips | 40x24mm or 60x24mm | |||
| Press-to-seal silicon insulator | Tool | Glace Bio-Labs | JTR20-2.5 | 2.5mm depth x 20mm diameter |
| NUNC Lab-Tek Chambered Coverglass | Tool | Fisher | 12-565-471 | 2wells/slides |
| Grid slide glass | Tool | or Finder graticule for cell counting |
Cite This Article
Ogata, S., Cho, K. W. Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay. J. Vis. Exp. (3), e187, doi:10.3791/187 (2007).