TITLE: Lanthanum DAB metals, Ln-DAB2 labeling of APEX2
AUTHORS: Stephen R. Adams, Mason R. Mackey, Alice Ting, Mark Ellisman, Thomas Deerinck

[DESCRIPTION]
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.

[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.


[STEPS]
1. HEK293T cells are cultured on imaging plates containing poly-d-lysine coated glass bottom No. 0 coverslips (P35GC-0-14C, MatTek Corporation).
2. 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.
3. 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.
4. Fixative is removed and cells are rinsed with 0.1M sodium cacodylate buffer, pH 7.4 (5X1min) on ice.
7. 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.
8.  After reactions, all plates of cells are rinsed with 0.1M sodium cacodylate buffer, pH 7.4 containing 2 mM CaCl2 (5X1min) on ice.
9. 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. 
10. Post fixative is removed from cells and are rinsed with 0.1M sodium cacodylate buffer at pH 7.4 (5X1min) on ice.
11. Cells are washed with ddH2O (5X1min) on ice.
12.  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.
5. Add 20mM glycine in 0.10M sodium cacodylate buffer, pH 7.4 for 15-20 minutes. 

6. Rinse cells 2x for 1 minute with the 0.10M sodium cacodylate buffer, pH 7.4. 
13. Cells were infiltrated with one part Durcupan ACM epoxy resin (44610, Sigma-Aldrich) to one part anhydrous ethanol for 30 minutes.
14. 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.
9.1. After 15 minutes, the 0.01% ruthenium tetroxide solution should be removed and replaced with new 0.01% ruthenium tetroxide solution.