TITLE: Labeling and DAB oxidation of EdU-treated HEK293T cells with Cy5 azide and Fe-TAML azide for light and transmission electron microscopy
AUTHORS: Stephen Adams, Mason Mackey, Mark H. Ellisman

[DESCRIPTION]
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]
1. HEK293T cells were plated onto MatTek dishes containing 35mm glass bottom No. 0 coverslips coated with poly-d-lysine.
2. The next day, 10 μM EdU is added to the cells and incubated for 720 min
3. 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 5 min  
minutes at 37 °Cand then at 4 °C  for 55 min . 

5. Wash cells with PBS pH 7.4 (2 x 1 min) at room temperature.
6. Rinse cells (2 x 1 min) with filtered 1% BSA in PBS pH 7.4 at room temperature.
7. 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).
 


8. After 30 minutes, a second 100 μl aliquot of newly prepared aqueous sodium ascorbate (100 mM) is added for another 30 min incubation.

9. Quickly quick rinse cells twice with filtered 1% BSA in PBS pH 7.4 at room temperature.
10. Wash cells with PBS pH 7.4 (5 x 1 min) at room temperature.
11. Collect fluorescence imaging of the labeled cells with Cy5 azide.
12. Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 (5 x 1 min).
13. 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.

14. 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 15 min and 30 min.

15. Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 (3 x 1 min).
16. Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 (5 x 1 min) at 4 °C.

17. 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 30 min at 4 °C.

18. Wash cells (5 x 2 min) with ddH2O at4 °C.

19. Dehydrate cells with an ice-cold graded dehydration ethanol series of 20%, 50%, 70%, 90%, 100% (anhydrous) for 1 min each and 3 x 100% (anhydrous) at room temperature for 1 min each.

20. Infiltrate cells with one-part Durcupan ACM epoxy resin (44610, Sigma-Aldrich) to one-part anhydrous ethanol for 30 min, 2 times with 100% Durcupan resin for 60 min each, a final change of Durcupan resin and immediately placed in a vacuum oven at 60 °C for 2880 minto harden.

4.3. Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 1 min at 4 °C. (3/5)
4.2. Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 1 min at 4 °C. (2/5)
4.1. Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 1 min  at 4 °C. (1/5)
4. 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
4.4. Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 1 min at 4 °C. (145)
4.5. Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 1 min at 4 °C. (5/5)
5.2. Wash cells with PBS pH 7.4 for 1 min   at room temperature. (2/2)
5.1. Wash cells with PBS pH 7.4 for 1 min  at room temperature. (1/2)
6.2. Rinse cells for 1 min   with filtered 1% BSA in PBS pH 7.4 at room temperature. (2/2)
6.1. Rinse cells for 1 min  with filtered 1% BSA in PBS pH 7.4 at room temperature. (1/2)

10.5. Wash cells with PBS pH 7.4 for 1 min at room temperature. (5/5)
10.4. Wash cells with PBS pH 7.4 for 1 min at room temperature. (4/5)
10.3. Wash cells with PBS pH 7.4 for 1 min at room temperature. (3/5)
10.2. Wash cells with PBS pH 7.4 for 1 min at room temperature. (2/5)
10.1. Wash cells with PBS pH 7.4 for 1 min at room temperature. (1/5)

12.5. Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 1 min. (5/5)
12.4. Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 1 min. (4/5)
12.3. Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 1 min. (3/5)
12.2. Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 1 min. (2/5)
12.1. Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 1 min. (1/5)

15.3. Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 1 min. (3/3)
15.2. Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 1 min. (2/3)
15.1. Wash cells with 100 mM NaCl 50 mM Na·Bicine pH 8.3 for 1 min. (1/3)

16.5. Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2  for 1 min at 4 °C. (5/5)
16.4. Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2  for 1 min at 4 °C. (4/5)
16.3. Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2  for 1 min at 4 °C. (3/5)
16.2. Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2  for 1 min at 4 °C. (2/5)
16.1. Wash cells with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 for 1 min at 4 °C. (1/5)

18.2. Wash cells for 2 min with ddH2O at 4 °C . (2/2)

18.1. Wash cells for 2 min with ddH2O at 4 °C . (1/2)