アフリカツメガエルの胚および細胞接着アッセイのアセンブリから主催し、動物極の外植片の解剖
Summary
自分の眉製の特殊な解剖ツール – このビデオでは、眉毛のナイフの使用を含むアフリカツメガエルの胚から主催者と動物極の外植片、の調製に用いられる技法を示しています。接着アッセイを組み立てるためのプロトコルも与え、その適切な開発のための重要な面の主催者や動物極細胞上に存在する主要な接着分子の存在のためのプローブです。
Protocol
準備接着チェンバース（注入/解剖前日） 接着アッセイチャンバーあなたが従来の複合顕微鏡を（ステージ上の光学位置）がある場合、それは低身長の付着室は、次のようにするためncecessaryです。倒立光学顕微鏡を使用している場合は、"NUNCのLab – Tek社チェンカバーガラスは、"単に使用され、ステップ＃1へスキップする – 2。 40 × 24ミリ…
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).