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.
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).