This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife – a specialized dissection tool made of one’s eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.
Preparing Adhesion Chambers (the day before the injection/dissection)
Adhesion Assay Chambers
If you have a conventional compound microscope (optical locates above the stage), it is ncecessary to make a low-height adhesion chamber as follows. If using the inverted-optics microscope, then “NUNC Lab-Tek Chambered Coverglass” can just be used, and skip the steps #1 – 2.
Coating the Chamber
Dissection and Dissociation of Animal Cap Explants
Cell Adhesion Assay
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 |