Ln-DAB2 Solutions
Mark Ellisman, Mason Mackey, Stephen R. Adams, Roger Tsien, Jeffrey D. Martell
Abstract
We use multicolor EM (electron microscopy) to paint multiple cellular markers by locally depositing specific Ln3+ from prepared solutions of Ln-DAB2 by mSOG, APEX2 or HRP. Each Ln3+ is then visualized by electron energy-loss spectroscopy and energy-filtered EM. Elemental maps are overlaid on conventional EM give multicolor EM.
Steps
To make 10 mL of a 2 mM Ln, Ce or Pr-DAB2 solution, 15.6 mg (20 μmol) of DTPA-DAB2 is suspended in 0.25 mL N,N Dimethylformamide (DMF) and gently heated to about 50C and sonicated/vortexed to dissolve.
8.33 mL of DDH2O is added to give a cloudy solution that cleared on addition of LnCl3 aqueous solution (0.1 M of LaCl3·6H2O, CeCl3·6H2O, or PrCl3·xH2O; the latter stock solution is dissolved in 0.1 M HCl) with 120 μL of La or Ce solutions or 140 μL of Pr solution, followed by vortexing and bath sonication to give clear light-brown solutions.
Aqueous NaOH solution (1 M) is added sequentially in six equal portions (6 × 10 μL) with vortexing after each addition.
(A precipitate is initially formed during the early steps of this neutralization but a mostly clear solution is present by the end).
1.67 mL of 0.3 M sodium cacodylate buffer, pH 7.4 is added, mixed, and centrifuged (3000 × g, 10 min) to remove any precipitate.
Solutions are syringe-filtered (0.22 μm, Millipore) immediately prior to addition to cells.
Metal ion concentrations can be measured by inductively coupled plasma mass spectroscopy (Agilent 7700).
