Sep 30, 2025

Public workspaceDAB precipitation by Laccase Oxidation 

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6195zvqe/v1
  • Song-Yi Lee1,
  • Heegwang Roh1,
  • Mason Mackey2,
  • Alice Y. Ting1,
  • Mark Ellisman2
  • 1Stanford University;
  • 2UCSD
  • NCMIR@UCSD
National Center for Microscopy and Imaging Research
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6195zvqe/v1
Protocol Citation: Song-Yi Lee, Heegwang Roh, Mason Mackey, Alice Y. Ting, Mark Ellisman 2025. DAB precipitation by Laccase Oxidation . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6195zvqe/v1
Manuscript citation:
Directed evolution of LaccID for cell surface proximity labeling and electron microscopy. Nature chemical biology Lee, S., Roh, H., Gonzalez-Perez, D., Mackey, M. R., Hoces, D., McLaughlin, C. N., Lin, C., Adams, S. R., Nguyen, K., Kim, K., Luginbuhl, D. J., Luo, L., Udeshi, N. D., Carr, S. A., Hernandez-Lopez, R. A., Ellisman, M. H., Alcalde, M., Ting, A. Y. 2025
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: February 23, 2022
Last Modified: September 30, 2025
Protocol Integer ID: 58672
Keywords: Oxidation, DAB precipitation, Laccase, dab precipitation by laccase oxidation, laccase oxidation, precipitates dab with oxygen, precipitates dab, encoded oxidase, dab precipitation, laccid, dab, oxidation, need for h2o2, h2o2, oxygen
Funders Acknowledgements:
National Science Foundation
Grant ID: UTA20-000889 to A.Y.T
National Science Foundation
Grant ID: 2014862 to M.H.E
Abstract
Genetically-encoded oxidase, LaccID, oxidizes and precipitates DAB with oxygen and therefore without the need for H2O2 or light. LaccID-expressing HEX293T cells precipitated DAB and were processed for TEM.
Materials
1. MatTek dishes containing 35mm glass bottom No. 0 coverslips coated with poly-d-lysine (P35GC-0-14C,
MatTek Corporation)

2. 0.1M sodium cacodylate with 2 mM CaCl2, pH 7.4 (12310, Electron Microscopy Sciences)

3. 2% glutaraldehyde (18426, Ted Pella Incorporated)

4. 3,3’- Diaminobenzidine (DAB) (D8001-10G, Sigma-Aldrich)

5. 1% osmium tetroxide (EMS, 19150)

6. Ethanol series

7. Durcupan ACM resin (Sigma-Aldrich, 44610)
Troubleshooting
Safety warnings
Use PPE for toxic chemicals.
Cell Fixation
LaccID-expressing HEX293T cells are plated onto MatTek dishes containing 35mm glass bottom No. 0 coverslips coated with poly-d-lysine (P35GC-0-14C, MatTek Corporation).
Cells are fixed using warm 2% glutaraldehyde (18426, Ted Pella Incorporated) in buffer 0.1M sodium cacodylate with 2 mM CaCl2, pH 7.4 (12310, Electron Microscopy Sciences) and then quickly moved to ice for 55 minutes.
Cells are washed five times for 2 min each on ice with cold 0.1M sodium cacodylate with 2 mM CaCl2, pH 7.4
Cell are blocked for 5 min with cold 20 mM glycine in 0.1M sodium cacodylate with 2 mM CaCl2, pH 7.4
A freshly solution of 5.4 mg of 3,3’- Diaminobenzidine (DAB) (D8001-10G, Sigma-Aldrich) is dissolved in 1.0 ml of 0.1 N HCl and added to 9.0 ml 0.1M sodium cacodylate buffer with 2 mM CaCl2, pH 7.4 buffer.
The DAB solution is added to cells for at least 3-30 min at room temperature until the desired brown intensity color of the precipitate was visible.
Cells are then washed five times for 2 min each with cold 0.1M sodium cacodylate with 2 mM CaCl2, pH 7.4.
TEM processing
Cells are post-fixed with 1% osmium tetroxide (EMS, 19150) in 0.1 M sodium cacodylate buffer pH 7.4 for 30 min on ice.
Postfixative is removed and cells are washed (five times for 1 min each) with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 on ice
Cells are washed (five times for 1 min each) with cold double-distilled H2O (ddH2O) on ice.
Cells are dehydrated with an ethanol series (20%, 50%, 70%, 90% and 100%) on ice for 1 min each.
Cells are dehydrated with 100% dry ethanol at room temperature three times for 1 min each.
Cells are infiltrated with 50:50 dry ethanol:Durcupan ACM resin (Sigma-Aldrich, 44610) for 30 min and then four changes of Durcupan at 1 h each and finally embedded in a vacuum oven at 60 °C for 72 hours.
Protocol references
Directed evolution of LaccID for cell surface proximity labeling and electron microscopy. Nature chemical biologyLee, S., Roh, H., Gonzalez-Perez, D., Mackey, M. R., Hoces, D., McLaughlin, C. N., Lin, C., Adams, S. R., Nguyen, K., Kim, K., Luginbuhl, D. J., Luo, L., Udeshi, N. D., Carr, S. A., Hernandez-Lopez, R. A., Ellisman, M. H., Alcalde, M., Ting, A. Y.2025
Acknowledgements
  1. Department of Genetics, Stanford University, Stanford, CA, USA Song-Yi Lee, Daniel Hoces, Chang Lin, Rogelio A. Hernández-López & Alice Y. Ting
  2. Department of New Biology, DGIST, Daegu, Republic of Korea Song-Yi Lee
  3. New Biology Research Center, DGIST, Daegu, Republic of Korea Song-Yi Lee
  4. Department of Chemistry, Stanford University, Stanford, CA, USA Heegwang Roh & Alice Y. Ting
  5. Department of Biology, Stanford University, Stanford, CA, USA Alice Y. Ting
  6. The Phil & Penny Knight Initiative for Brain Resilience at the Wu Tsai Neurosciences Institute, Stanford, CA, USA Alice Y. Ting
  7. Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA Mason R. Mackey, Keun-Young Kim & Mark H. Ellisman
  8. Center for Research in Biological Systems and the National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA, USA Mason R. Mackey & Mark H. Ellisman
3. Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA
Stephen Adams