Labeling and DAB oxidation of EdU-treated HEK293T cells with Cy5 azide and Fe-TAML azide for light and transmission electron microscopy
Mark H. Ellisman, Mason Mackey, Stephen Adams
HEK293T cells
labeling
oxidation
Cy5 azide
Fe-TAML azide
light microscopy
transmission electron microscopy
fixation
click chemistry
DAB2 oxidation
embedding
microscopy analysis
Fe-TAML
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.
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.
Steps
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 12h 0m 0s
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 0h 5m 0s
minutes at 37°Cand then at 4°C for 0h 55m 0s .
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 0h 1m 0s at 4°C. (1/5)
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 0h 1m 0s at 4°C. (2/5)
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 0h 1m 0s at 4°C. (3/5)
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 0h 1m 0s at 4°C. (145)
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 0h 1m 0s at 4°C. (5/5)
Wash cells with PBS pH 7.4 (2 x 1 min) at room temperature.
Wash cells with PBS pH 7.4 for 0h 1m 0s at room temperature. (1/2)
Wash cells with PBS pH 7.4 for 0h 1m 0s at room temperature. (2/2)
Rinse cells (2 x 1 min) with filtered 1% BSA in PBS pH 7.4 at room temperature.
Rinse cells for 0h 1m 0s with filtered 1% BSA in PBS pH 7.4 at room temperature. (1/2)
Rinse cells for 0h 1m 0s with filtered 1% BSA in PBS pH 7.4 at room temperature. (2/2)
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 0h 30m 0s incubation.
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 0h 1m 0s at room temperature. (1/5)
Wash cells with PBS pH 7.4 for 0h 1m 0s at room temperature. (2/5)
Wash cells with PBS pH 7.4 for 0h 1m 0s at room temperature. (3/5)
Wash cells with PBS pH 7.4 for 0h 1m 0s at room temperature. (4/5)
Wash cells with PBS pH 7.4 for 0h 1m 0s at room temperature. (5/5)
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 0h 1m 0s. (1/5)
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 0h 1m 0s. (2/5)
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 0h 1m 0s. (3/5)
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 0h 1m 0s. (4/5)
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 0h 1m 0s. (5/5)
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 0h 15m 0s and 0h 30m 0s.
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 0h 1m 0s. (1/3)
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 0h 1m 0s. (2/3)
Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 0h 1m 0s. (3/3)
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 0h 1m 0s at 4°C. (1/5)
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 0h 1m 0s at 4°C. (2/5)
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 0h 1m 0s at 4°C. (3/5)
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 0h 1m 0s at 4°C. (4/5)
Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 0h 1m 0s at 4°C. (5/5)
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 0h 30m 0s at 4°C.
Wash cells (5 x 2 min) with ddH2O at4°C.
Wash cells for 0h 2m 0s with ddH2O at 4°C . (1/2)
Wash cells for 0h 2m 0s with ddH2O at 4°C . (2/2)
Dehydrate cells with an ice-cold graded dehydration ethanol series of 20%, 50%, 70%, 90%, 100% (anhydrous) for 0h 1m 0s each and 3 x 100% (anhydrous) at room temperature for 0h 1m 0s each.
Infiltrate cells with one-part Durcupan ACM epoxy resin (44610, Sigma-Aldrich) to one-part anhydrous ethanol for 0h 30m 0s, 2 times with 100% Durcupan resin for 1h 0m 0s each, a final change of Durcupan resin and immediately placed in a vacuum oven at 60°C for 48h 0m 0sto harden.
