Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

Summary

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.

Protocol

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.

1. Attach one of "press-to-seal silicon insulator" on a 40 x 24mm cover slip. Firmly press the insulator to the cover slip on clean benchtop and look from the other side to make sure that the insulator is completely attached without any air....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x MBS-H	Buffer			Modified Barth’s medium: 1 L recipe: 5.14 g NaCl7 5 mg KCl 0.2 g NaHCO3 0.2 g MgSO4•7H2O 60 mg CaCl2•2H2O 80 mg Ca(NO3)2•4H2O2. 38 g HEPES, free-acidAdjust the pH to 7.4 with NaOH. Autoclave.
CMF-MBS	Buffer			This is Ca2+/Mg2+-free version of MBS-H. 500 mL recipe: 2.57 g NaCl 37.5 mg KCl 0.1 g NaHCO3/sub> 1.19 g 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-Aldrich	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 –20 °C. 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-Aldrich	F-1141	This pre-made solution can be used as 50x stock. Make 1/50 dilution with 1x MBS-H make 20 μg/mL working solution just before you use. You need 200 μL 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				40 mm x 24 mm or 60 mm x 24 mm
Press-to-seal silicon insulator	Tool	Glace Bio-Labs	JTR20-2.5	2.5 mm depth x 20 mm diameter
NUNC Lab-Tek Chambered Coverglass	Tool	Fisher Scientific	12-565-471	2 wells/slides
Grid slide glass	Tool			or Finder graticule for cell counting