Sep 16, 2022
Ln-DAB2 Solutions
- Stephen R. Adams1,2,
- Mason Mackey2,
- Roger Tsien1,3,4,
- Mark Ellisman2,5,
- Jeffrey D. Martell6
- 1Department of Pharmacology, University of California, San Diego, La Jolla CA;
- 2National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla CA;
- 3Howard Hughes Medical Institute, University of California, San Diego, La Jolla CA;
- 4Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla CA;
- 5Department of Neurosciences, University of California, San Diego, La Jolla CA;
- 6Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
- NCMIR@UCSD
Protocol Citation: Stephen R. Adams, Mason Mackey, Roger Tsien, Mark Ellisman, Jeffrey D. Martell 2022. Ln-DAB2 Solutions. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj6zdblk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working.
Created: May 11, 2022
Last Modified: September 16, 2022
Protocol Integer ID: 62439
Keywords: Ln-DAB2 solution, multiple cellular marker, specific ln3, electron microscopy, depositing specific ln3, dab2 solution, multicolor em, ln3, dab2 by msog, solutions of ln, elemental map, dab2, loss spectroscopy, ln
Funders Acknowledgements:
UCSD Graduate Training Programs in Cellular and Molecular Pharmacology
Grant ID: T32 GM007752
Neuroplasticity of Aging
Grant ID: T32 AG000216
NIH
Grant ID: GM103412
NIH
Grant ID: GM086197
4D Stem Grant
Grant ID: R01GM138780
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.
Guidelines
Ln-DAB2 solutions in cacodylate buffer were prepared immediately before use at room temperature.
Note: This buffer is hazardous and any waste should be handled accordingly.
Safety warnings
Wear PPE.
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).