Jun 06, 2024
Labeling and DAB oxidation of EdU-treated HEK293T cells with Cy5 azide and Fe-TAML azide for light and transmission electron microscopy
- Stephen Adams1,2,
- Mason Mackey1,2,
- Mark H. Ellisman1,2
- 1UC San Diego Health Sciences;
- 2National Center for Microscopy and Imaging Research
- NCMIR@UCSD
External link: https://www.biorxiv.org/content/10.1101/2023.08.25.554352v2
Protocol Citation: Stephen Adams, Mason Mackey, Mark H. Ellisman 2024. Labeling and DAB oxidation of EdU-treated HEK293T cells with Cy5 azide and Fe-TAML azide for light and transmission electron microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjn8jxgk5/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 16, 2024
Last Modified: June 06, 2024
Protocol Integer ID: 99987
Keywords: HEK293T cells, labeling, oxidation, Cy5 azide, Fe-TAML azide, light microscopy, transmission electron microscopy, fixation, click chemistry, DAB2 oxidation, embedding, microscopy analysis, Fe-TAML
Funders Acknowledgements:
R01
Grant ID: GM138780 (MHE)
R01
Grant ID: GM086197 (SRA/DB)
R35
Grant ID: GM128859 (JTN)
NSF
Grant ID: 2014862 (MHE)
R01
Grant ID: AG081037 (MHE)
Disclaimer
This protocol is provided for informational purposes only and should be performed by individuals trained in laboratory techniques and safety procedures. The authors and publishers of this protocol do not assume any responsibility for accidents or damage resulting from the use of this protocol. Users are encouraged to consult additional references and relevant safety data sheets for specific reagents and procedures employed in this protocol.
Abstract
This protocol outlines the click chemistry labeling of EdU-pulsed DNA with Fe-TAML azide to catalyze DAB oxidation by hydrogen peroxide to generate localized osmiophilic precipitate detectable by light and electron microscopy.
Materials
Click Buffer
| A | B | |
| 0.1M NaCl | 0.29 g | |
| 50 mM HEPES pH 7.4 | 0.595 g | |
| 0.1% Saponin | 50 mg | |
| Double Distilled Water | 50 mL | |
| 1N Sodium Hydroxide | 1.25 mL |
Before start
Prior to implementing this protocol, ensure all materials and reagents are prepared according to the specified concentrations and conditions. Adhere strictly to the indicated time frames and temperatures during each step to achieve optimal results. Perform all procedures in a designated laboratory space equipped with appropriate safety measures and waste disposal systems.
HEK293T cells were plated onto MatTek dishes containing 35mm glass bottom No. 0 coverslips coated with poly-d-lysine.
The next day, 10 μM EdU is added to the cells and incubated for 12:00:00
12h
Cells are fixed with 2% glutaraldehyde (16220, Electron Microscopy Sciences) in 0.1 M sodium cacodylate buffer (18851, Ted Pella), pH 7.4 containing 2 mM CaCl2 for 00:05:00
minutes at 37 °C and then at 4 °C for 00:55:00 .
1h
Remove fixative and wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 (5 x 1 min) at 4 °C
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 00:01:00 at 4 °C . (1/5)
1m
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 00:01:00 at 4 °C . (2/5)
1m
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 00:01:00 at 4 °C . (3/5)
1m
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 00:01:00 at 4 °C . (145)
1m
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 00:01:00 at 4 °C . (5/5)
1m
Wash cells with PBS pH 7.4 (2 x 1 min) at room temperature.
Wash cells with PBS pH 7.4 for 00:01:00 at room temperature. (1/2)
1m
Wash cells with PBS pH 7.4 for 00:01:00 at room temperature. (2/2)
1m
Rinse cells (2 x 1 min) with filtered 1% BSA in PBS pH 7.4 at room temperature.
Rinse cells for 00:01:00 with filtered 1% BSA in PBS pH 7.4 at room temperature. (1/2)
1m
Rinse cells for 00:01:00 with filtered 1% BSA in PBS pH 7.4 at room temperature. (2/2)
1m
Carry out the Click reaction of the cells at room temperature and protect them from light. Use a mixture of 1.0 μM Cy5 azide and 28 μM Fe-TAML azide solution freshly prepared from 900 μl click buffer, 10.0 μl CuSO4 (100 mM in water), and the reaction initiated with 100 𝜇l of freshly prepared aqueous sodium ascorbate (100 mM).
| A | B | |
| 10 mM Cy5 azide | 1.0 μl | |
| 28 μM Fe-TAML azide | 1.0 μl | |
| Click buffer (see materials) | 900 μl | |
| 100 mM CuSO4 | 10.0 μl | |
| 100 mM aqueous sodium ascorbate | 100 μl |
After 30 minutes, a second 100 μl aliquot of newly prepared aqueous sodium ascorbate (100 mM) is added for another 00:30:00 incubation.
30m
Quickly quick rinse cells twice with filtered 1% BSA in PBS pH 7.4 at room temperature.
Wash cells with PBS pH 7.4 (5 x 1 min) at room temperature.
Wash cells with PBS pH 7.4 for 00:01:00 at room temperature. (1/5)
1m
Wash cells with PBS pH 7.4 for 00:01:00 at room temperature. (2/5)
1m
Wash cells with PBS pH 7.4 for 00:01:00 at room temperature. (3/5)
1m
Wash cells with PBS pH 7.4 for 00:01:00 at room temperature. (4/5)
1m
Wash cells with PBS pH 7.4 for 00:01:00 at room temperature. (5/5)
1m
Collect fluorescence imaging of the labeled cells with Cy5 azide.
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 (5 x 1 min).
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 00:01:00 . (1/5)
1m
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 00:01:00 . (2/5)
1m
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 00:01:00 . (3/5)
1m
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 00:01:00 . (4/5)
1m
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 00:01:00 . (5/5)
1m
5.4 mg of 3,3’- Diaminobenzidine (DAB) (D8001-10G, Sigma-Aldrich) is dissolved in 1.0 ml of 0.1 N HCl and 9.0 ml of 50 mM Bicine 100 mM NaCl pH 8.3 was added with 10 μl H2O2 (final, 40 mM from 30% stock) to the DAB solution.
Add the DAB/H2O2 solution to the cells by a 0.22μm Millex 33mm PES sterile filter (SLGSR33RS, Sigma-Aldrich) at room temperature. Reaction time is between 00:15:00 and 00:30:00 .
45m
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 (3 x 1 min).
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 00:01:00 . (1/3)
1m
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 00:01:00 . (2/3)
1m
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 00:01:00 . (3/3)
1m
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 (5 x 1 min) at 4 °C .
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 00:01:00 at 4 °C . (1/5)
1m
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 00:01:00 at 4 °C . (2/5)
1m
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 00:01:00 at 4 °C . (3/5)
1m
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 00:01:00 at 4 °C . (4/5)
1m
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 00:01:00 at 4 °C . (5/5)
1m
Post fix cells with 1% osmium tetroxide (19150, Electron Microscopy Sciences) containing 0.8% potassium ferrocyanide and 2 mM CaCl2 in 0.1 M sodium cacodylate buffer pH 7.4 for 00:30:00 at 4 °C .
30m
Wash cells (5 x 2 min) with ddH2O at4 °C .
Wash cells for 00:02:00 with ddH2O at 4 °C . (1/2)
2m
Wash cells for 00:02:00 with ddH2O at 4 °C . (2/2)
2m
Dehydrate cells with an ice-cold graded dehydration ethanol series of 20%, 50%, 70%, 90%, 100% (anhydrous) for 00:01:00 each and 3 x 100% (anhydrous) at room temperature for 00:01:00 each.
2m
Infiltrate cells with one-part Durcupan ACM epoxy resin (44610, Sigma-Aldrich) to one-part anhydrous ethanol for 00:30:00 , 2 times with 100% Durcupan resin for 01:00:00 each, a final change of Durcupan resin and immediately placed in a vacuum oven at 60 °C for 48:00:00 to harden.
2d 1h 30m
Protocol references
Adams, Stephen R., et al. "Fe-TAMLs as a new class of small molecule peroxidase probes for correlated light and electron microscopy." bioRxiv (2023): 2023-08.