Sep 30, 2025

Public workspaceTwo Color TEM Labeling using Silver-Gold Intensification of DAB and CuDAB2

DOI
https://dx.doi.org/10.17504/protocols.io.261ge5beog47/v1
  • Mason Mackey1,
  • Stephen Adams1,
  • Mark Ellisman1
  • 1UCSD
  • NCMIR@UCSD
National Center for Microscopy and Imaging Research
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DOI: https://dx.doi.org/10.17504/protocols.io.261ge5beog47/v1
Protocol Citation: Mason Mackey, Stephen Adams, Mark Ellisman 2025. Two Color TEM Labeling using Silver-Gold Intensification of DAB and CuDAB2. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5beog47/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: September 19, 2024
Last Modified: September 30, 2025
Protocol Integer ID: 107930
Keywords: Photooxidation, oxidation, miniSOG-mitomatrix, Fe-TAML, Gold enhancement, Two color, CuDAB2, color labeling of cell organelle, color tem labeling, thermofisher talos 200c g2 transmission electron microscope, gold intensification on polymerized dab, color labeling, gold intensification of dab, using silver, dna from oxidation, polymerized dab, cell organelle, labeling, cudab2 this method, photooxidation of minisog, photooxidation, cudab2, silver
Funders Acknowledgements:
NIH
Grant ID: 1R01GM138780
Abstract
This method is use for two color labeling of cell organelles for transmission electro microscopy. The color TEM labeling used Silver-Gold Intensification on polymerized DAB from photooxidation of miniSOG-mitomatrix and CuDAB2 labeling on DNA from oxidation of FeTAML-azide. The labeling were confirmed by EELS using a Gatan Imaging Filter on a ThermoFisher Talos 200C G2 transmission electron microscope at 200kV.
Guidelines
The method is use for two color labeling of cell organelles for transmission electro microscopy. 80 um sections were collected and metals were verified using EELS from a Gatan Imaging Filter (GIF).
Materials
1. HEK293T cells 
2. MatTek dishes containing 35mm glass bottom No. 0 coverslips coated with poly-d-lysine (P35GC-0-14C, 3.
MatTek Corporation)
3. EdU Quantity: 50 mg (A10044, ThermoFisher)
4. 100 mM NaCl 50 mM bicine pH 8.3 
5. 2 mM Cu-DAB2 
6. 2% EM grade glutaraldehyde (18426, Ted Pella Incorporated)
7. 1X PBS
8. FeTAML-azide
9. 2% sodium acetate
10. Heating plate
11. 2.5% sodium thiosulphate
12. 5% silver nitrate
13. 0.05% tetrachlorogold (III) acid trihydrate 
14. 3% hexamethylenetetramine
15. 2.5% disodium tetraborate
16. Lipofectamine 3000 (Life Technologies)
17. standard GFPex475 filter set
18. 1X PBS
Troubleshooting
Safety warnings
Use PPE to protect from toxic chemicals.
Fixation and photooxidation of miniSOG-Mitomatrix.
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 miniSOG-mitomatrix fusion using Lipofectamine 3000 (Life Technologies). MiniSOG is fused to N-terminal of mitomatrix. Cells also are incubated with 10 μM EdU for 12hours. 
Fix cells with 2% EM grade glutaraldehyde (18426, Ted Pella Incorporated) in 1XPBS, pH 7.2 (18851, Ted Pella Incorporated) containing for 5 minutes at 37oC and then on ice for 55 minutes.
*Rinse once with fix after removing cell culture media. Afterwards add the fix for the
total duration.
Wash 5X with 1X PBS buffer pH 7.2 for 2 minutes each on ice.
Blocking step for miniSOG: Block solution: (20 mM glycine, 10 mM KCN and 10 mM aminotriazole).
Cells are blocked for 20 minutes on ice.
Wash 2X with 1X PBS, pH 7.2 for 2 minutes each on ice.
For mSOG, regions of interest are identified by standard GFPex475 filter set at light intensity of 10% and for photooxidation of DAB in 1X PBS at pH 7.2 the light intensity is set at 100%. A photooxidation reaction with DAB in 1X PBS at pH 7.2 until the desired brown intensity color from the precipitate for 2-5 minutes.
Wash 5X with 1X PBS, pH 7.2 for 2 minutes each on ice.
Gold enhancement on polymerized DAB.
Wash 3X with 2% sodium acetate for 1 minutes each at room temperature.
Cells in MatTek plate are prewarmed at 60°C for 10 min in 2% sodium acetate.
Make enhancement solutions containing 3% hexamethylenetetramine in ddH2O, 5% silver nitrate in ddH2O, and 2.5% disodium tetraborate in ddH2O, mixed in a ratio of 20:1:2, prewarm solution for 10 minutes at 60oC, added to the cells and  incubated for 8-11 min at 60°C.
Wash 5X3 min with 2% sodium acetate at room temperature.
Incubate cells with 0.05% tetrachlorogold (III) acid trihydrate in ddH2O for 5 minutes at room temperature.
Wash 5X3 min with 2% sodium acetate at room temperature.
Incubated with 2.5% sodium thiosulphate in ddH2O for 4 min at room temperature.
Wash 1X3 min with 2% sodium acetate room temperature.
Wash 5X1 min with 1XPBS on ice.
Click reaction of Fe-TAML-azide to EdU
The click reaction of the cells were carried out at room temperature and protected from light by using a mixture of 28 μM Fe-TAML azide solution, freshly prepared from 900 μl click buffer (100 mM NaCl 50 mM Na·HEPES pH 7.6), 10.0 μl CuSO4 (100 mM in water), 0.1% Saponin (Sigma, S-4521) and the reaction was initiated with 100 𝜇l of freshly prepared aqueous sodium ascorbate (100 mM).
After 30 minutes, a second 100 𝜇L aliquot of newly prepared aqueous sodium ascorbate (100 mM) was added for another 30 minutes incubation.
Cells are washed with PBS pH 7.4 (5 x 1 min) at room temperature.
Cells are washed with 100 mM NaCl 50 mM Na·Bicine pH 8.3 ( 5 x 1 min).
Copper-DAB polymerization
2 mM Cu-DAB2 solutions in 100 mM NaCl 50 mM bicine pH 8.3 were prepared similarly to previously reported solutions of Ln-DAB2 in cacodylate buffer (Adams et al 2016) by substituting with stock solutions of 0.1M CuSO4, 1M-bicine buffer pH 8.3 and 1M-NaCl.
10 μl of H2O2  from a 30% stock solution was added to 10ml of the 2 mM Cu-DAB2.
The Cu-DAB2 reaction ran for one hour on the cells.
Cells are washed with 100 mM NaCl 50 mM Na·Bicine pH 8.3 ( 5 x 1 min).
Process for TEM.
Protocol references
1. Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species. Cell Chem Biol. 2016 Nov 17; 23(11):1417-1427. Adams SR, Mackey MR, Ramachandra R, Palida Lemieux SF, Steinbach P, Bushong EA, Butko MT, Giepmans BNG, Ellisman MH, Tsien RY. PMID: 27818300; PMCID: PMC5127401.


2. Fe-TAMLs as a new class of small molecule peroxidase probes for correlated light and electron microscopy. bioRxiv. 2023 Oct 04. Adams SR, Mackey MR, Ramachandra R, Deerinck TJ, Castillon GA, Phan S, Hu J, Boassa D, Ngo JT, Ellisman MH. PMID: 37662194; PMCID: PMC10473768.

3. A Simple Silver–Gold Intensification Procedure for Double DAB Labeling Studies in Electron Microscopy Rebecca Teclemariam–Mesbah, Joke Wortel, Herms J. Romijn, and Ruud M. Buijs. The Journal of Histochemistry & Cytochemistry. Volume 45(4): 619–621, 1997.

Acknowledgements
Steve Peltier
Mia Flores
Isabella Ramos