Sep 16, 2022
Lanthanum DAB metals, Ln-DAB2 labeling of APEX2
- Stephen R. Adams1,2,
- Mason Mackey2,
- Alice Ting3,4,5,6,
- Mark Ellisman2,7,8,
- Thomas Deerinck2
- 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;
- 3Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA;
- 4Department of Genetics, Stanford University, Stanford, California, USA;
- 5Department of Biology, Stanford University, Stanford, California, USA;
- 6Department of Chemistry, Stanford University, Stanford, California, USA;
- 7Department of Neurosciences, University of California at San Diego, La Jolla, California, USA;
- 8Department of Bioengineering, University of California at San Diego, La Jolla, California, USA
Protocol Citation: Stephen R. Adams, Mason Mackey, Alice Ting, Mark Ellisman, Thomas Deerinck 2022. Lanthanum DAB metals, Ln-DAB2 labeling of APEX2. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwkoe2vmk/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: 62438
Keywords: lanthanium, DAB metals, APEX2, HEK293T cells
Funders Acknowledgement:
UCSD Graduate Training Programs in Cellular and Molecular Pharmacology
Grant ID: (T32 GM007752)(SP)
Neuroplasticity of Aging
Grant ID: (T32 AG000216)(SP)
NIH
Grant ID: R24GM137200
NIH
Grant ID: P41 GM103412
NIH
Grant ID: R01GM086197
4D Stem Grant
Grant ID: R01GM138780
Abstract
This protocol uses APEX2, an engineered peroxidase that genetically targets a cellular region of interest, to oxidatively polymerize Ln-DAB2, (lanthanum chelates conjugated to DAB), using hydrogen peroxide. We use these precipitated lanthanum metals to identify the labeled regions of interest by using EELS and elemental mapping.
Safety warnings
Wear PPE.
Before start
Buffer Recipe: (65.7 ml of DDH2O + 1ml of 0.204 M CaCl2 + 33.3ml of 0.3M sodium cacodylate pH 7.4)
Fixative in conical tube: 2.0 ml from 25% EM grade glutaraldehyde stock vial, 1.0 ml of 0.3M stock sodium cacodylate buffer and 22.0 ml of 0.1M sodium cacodylate buffer, pH 7.4.
HEK293T cells are cultured on imaging plates containing poly-d-lysine coated glass bottom No. 0 coverslips (P35GC-0-14C, MatTek Corporation).
Cells are transiently transfected with either APEX2-H2B or mitochondrial matrix-APEX2 fusion using Lipofectamine 3000 (Life Technologies). APEX2 is fused to N-terminal of H2B and to C- terminal of mito matrix.
After 16 hours transfection, cells are fixed with 2% EM grade glutaraldehyde (18426, Ted Pella Incorporated) in 0.1M sodium cacodylate buffer, pH 7.4 (18851, Ted Pella Incorporated) containing 2 mM CaCl2 for 5 minutes at 37°C and then on ice for 55 minutes.
Fixative is removed and cells are rinsed with 0.1M sodium cacodylate buffer, pH 7.4 (5X1min) on ice.
Add 20mM glycine in 0.10M sodium cacodylate buffer, pH 7.4 for 15-20 minutes.
Rinse cells 2x for 1 minute with the 0.10M sodium cacodylate buffer, pH 7.4.
On a set plates, an enzymatic reaction with Ln-DAB2 with 4 mM H2O2 (from 30%) in 0.1M sodium cacodylate buffer at pH 7.4 until the desired brown intensity color from the precipitate, between 2 to 30 minutes. (1ul of 9.8M H2O2 added in 2500 ul or 2.5 ml Ln-DAB2 solution). See making of Ln-DAB2 solutions.
After reactions, all plates of cells are rinsed with 0.1M sodium cacodylate buffer, pH 7.4 containing 2 mM CaCl2 (5X1min) on ice.
Cells are post-fixed with either 0.01% ruthenium tetroxide (20700-05, Electron Microscopy Sciences) or 1% reduced osmium tetroxide (19150, Electron Microscopy Sciences) containing 2 mM CaCl2 and 0.8% potassium ferrocyanide in 0.1M sodium cacodylate buffer, pH 7.4 for 30 minutes.
After 15 minutes, the 0.01% ruthenium tetroxide solution should be removed and replaced with new 0.01% ruthenium tetroxide solution.
Post fixative is removed from cells and are rinsed with 0.1M sodium cacodylate buffer at pH 7.4 (5X1min) on ice.
Cells are washed with ddH2O (5X1min) on ice.
An ice-cold graded dehydration ethanol series of 20%, 50%, 70%, 90%, 100% (anhydrous) for one minute each and 2X 100% (anhydrous) at room temperature for 1 minute each.
Cells were infiltrated with one part Durcupan ACM epoxy resin (44610, Sigma-Aldrich) to one part anhydrous ethanol for 30 minutes.
Plates are changed three times with 100% Durcupan resin for 1 hours each with the first change having the lid only partly covering the top of the plate, a final change of Durcupan resin and immediately placing the plate without the lid in a vacuum oven at 60°C for 48 hours to harden.