TITLE: Labeling of microtubules using mouse anti-β-tubulin primary monoclonal antibody with secondary Fe-TAML-peg4-Cy5-goat anti-mouse IgG conjugate and oxidation of DAB with H2O2 for light and transmission electron microscopy
AUTHORS: Steven Adams, Mason R. Mackey, Mark Ellisman

[DESCRIPTION]
This protocol details the labeling of microtubules using mouse anti-β-tubulin primary monoclonal antibody with secondary Fe-TAML-peg4-Cy5-goat anti-mouse IgG conjugate and oxidation of DAB with H2O2 for light and transmission electron microscopy.

[STEPS]
SECTION: Labeling of microtubules
1. Culture HEK293T cells on MatTek plates containing poly-D-lysine coated glass bottom No.0 coverslips in DMEM supplement with 10% fetal bovine serum.
SECTION: Labeling of microtubules
2. Rinse the cells (x3) with cytoskeleton stabilizing buffer, at 37 °C  and fixed with 4% paraformaldehyde (19202, Electron Microscopy Sciences) and 0.05% glutaraldehyde (16220, Electron Microscopy Sciences) in CSB at 37 °C  for  5 min  and for 25 min  at  4 °C.

CSB buffer:
 
 
SECTION: Labeling of microtubules
3. After fixation, first wash the cells with CSB (5 x 1 min) at 4 °C. 

SECTION: Labeling of microtubules
5. Wash the cells in CSB buffer with 0.05% glycine (3 x 1 min) at 4°C.
SECTION: Labeling of microtubules
5.1. Wash the cells in CSB buffer with 0.05% glycine 1 min at  4 °C. (1/3)

SECTION: Labeling of microtubules
5.2. Wash the cells in CSB buffer with 0.05% glycine 1 min at  4 °C. (2/3)
SECTION: Labeling of microtubules
5.3. Wash the cells in CSB buffer with 0.05% glycine 1 min at  4 °C. (3/3)
SECTION: Labeling of microtubules
6. Block the cells with 1% BSA (A8022-100G, Sigma), 1% normal goat serum (NGS) and 0.05% glycine in CSB for 20 min at 4 °C. 

SECTION: Labeling of microtubules
7. Incubate the cells with primary mouse monoclonal antibody to β-tubulin (300-fold dilution, clone Tub2.1, T5201, Sigma) for 180 min  at  4 °C in 1% BSA, 1% NGS and 0.05% glycine in CSB buffer.

SECTION: Labeling of microtubules
8. Remove primary antibody and wash with 1% BSA, 1% NGS and 0.05% glycine in CSB (5 x 3 min) at 4°C.
SECTION: Labeling of microtubules
8.1. Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 3 min at 4 °C. (1/5)

SECTION: Labeling of microtubules
8.2. Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 3 min at 4 °C. (2/5)
SECTION: Labeling of microtubules
8.3. Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 3 min at 4 °C. (3/5)
SECTION: Labeling of microtubules
9. Incubate the cells with secondary Fe-TAML-peg4-Cy5-goat anti-mouse IgG conjugate in 1% BSA (0.15 ml diluted to 1ml) in 1% NGS and 0.05% glycine in CSB for 3 min at 4 °C. [Adams et al., 2023].

SECTION: Labeling of microtubules
10. Then wash the cells (5 x 1 min) with CSB at 4°C.
SECTION: Labeling of microtubules
10.1. Wash the cells for  1 min with CSB at  4 °C. (1/5)

SECTION: Labeling of microtubules
10.2. Wash the cells for  1 min with CSB at  4 °C. (2/5)
SECTION: Labeling of microtubules
10.3. Wash the cells for  1 min with CSB at  4 °C. (3/5)
SECTION: Labeling of microtubules
10.4. Wash the cells for  1 min with CSB at  4 °C. (4/5)
SECTION: Labeling of microtubules
10.5. Wash the cells for  1 min with CSB at  4 °C. (5/5)
SECTION: Labeling of microtubules
11. Fix the cells with 2% glutaraldehyde in CSB for 20 min  at 4 °C.

SECTION: Labeling of microtubules
12. Wash the cells (5 x 1 min) with CSB at 4°C. 
SECTION: Labeling of microtubules
12.1. Wash the cells for 1 min with CSB at 4 °C. (1/5)

SECTION: Labeling of microtubules
12.2. Wash the cells for 1 min with CSB at 4 °C. (2/5)
SECTION: Labeling of microtubules
12.3. Wash the cells for 1 min with CSB at 4 °C. (3/5)
SECTION: Labeling of microtubules
12.4. Wash the cells for 1 min with CSB at 4 °C. (4/5)
SECTION: Labeling of microtubules
12.5. Wash the cells for 1 min with CSB at 4 °C. (5/5)
SECTION: Labeling of microtubules
13. Image the cells for fluorescence labeling.
SECTION: Labeling of microtubules
14. Dissolve and add  5.4 mg of 3,3’- Diaminobenzidine (DAB) (D8001-10G, Sigma-Aldrich) in 1.0 mL  of 0.1 3261  HCl and 9.0 mL  of  50 millimolar (mM) Bicine 100 millimolar (mM) NaCl pH 8.3  with 10 µL H2O2 (final, 40 millimolar (mM) from 30% stock) to the DAB solution.

SECTION: Labeling of microtubules
15. Wash the cells (2 x 2 min) with 50 mM Bicine 100 mM NaCl pH 8.3 at 4°C.
SECTION: Labeling of microtubules
15.1. Wash the cells  for 2 min with 50 millimolar (mM) Bicine 100 millimolar (mM)NaCl pH 8.3 at 4 °C. (1/2)

SECTION: Labeling of microtubules
15.2. Wash the cells  for 2 min with 50 millimolar (mM) Bicine 100 millimolar (mM)NaCl pH 8.3 at 4 °C. (2/2)

SECTION: Labeling of microtubules
16. 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 60 min-90 min.

SECTION: Labeling of microtubules
17. Remove the DAB solution, and wash the cells with 50 mM Bicine 100mM NaCl pH 8.3 (2 x 2 min) on ice.
SECTION: Labeling of microtubules
17.1. Wash the cells with 50 millimolar (mM) Bicine 100 millimolar (mM) NaCl pH 8.3 for 2 min on ice . (1/2)

SECTION: Labeling of microtubules
17.2. Wash the cells with 50 millimolar (mM) Bicine 100 millimolar (mM) NaCl pH 8.3 for 2 min on ice . (2/2)

SECTION: Labeling of microtubules
18.1. Wash the cells with 0.1 Molarity (M)sodium cacodylate buffer pH 7.4 for 2 min. (1/3)

SECTION: Labeling of microtubules
18.2. Wash the cells with 0.1 Molarity (M)sodium cacodylate buffer pH 7.4 for 2 min. (2/3)

SECTION: Labeling of microtubules
18.3. Wash the cells with 0.1 Molarity (M)sodium cacodylate buffer pH 7.4 for 2 min. (3/3)

SECTION: Labeling of microtubules
19. Do a final primary fixation with 2% glutaraldehyde in 2 millimolar (mM) CaCl2 0.1 Molarity (M) sodium cacodylate pH 7.4 for 30 min at 4 °C.

SECTION: Labeling of microtubules
8.4. Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 3 min at 4 °C. (4/5)
SECTION: Labeling of microtubules
8.5. Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 3 min at 4 °C. (5/5)
SECTION: Labeling of microtubules
20. Remove the fixative and wash the cells (5 x 2 min) with 0.1 M sodium cacodylate (18851, Ted Pella) pH 7.4 at 4°C.

SECTION: Labeling of microtubules
21. All cells are post-fixed with 1% osmium tetroxide (19150, Electron Microscopy Sciences) containing 0.8% potassium ferrocyanide, 2 millimolar (mM) CaCl2 and in 0.1 Molarity (M) sodium cacodylate pH 7.4 for 30 min at 4 °C.

SECTION: Labeling of microtubules
22. Wash the cells (5 x 2 min) with ddH2O at 4°C.
SECTION: Labeling of microtubules
23. Dehydrate the cells by 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.

SECTION: Labeling of microtubules
22.1. Wash the cells for 2 min with ddH2O at 4 °C. (1/5)

SECTION: Labeling of microtubules
22.2. Wash the cells for 2 min with ddH2O at 4 °C. (2/5)
SECTION: Labeling of microtubules
22.3. Wash the cells for 2 min with ddH2O at 4 °C. (3/5)
SECTION: Labeling of microtubules
22.4. Wash the cells for 2 min with ddH2O at 4 °C. (4/5)
SECTION: Labeling of microtubules
22.5. Wash the cells for 2 min with ddH2O at 4 °C. (5/5)
SECTION: Labeling of microtubules
20.1. Wash the cells for 2 min with 0.1 Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4 °C. (1/5)

SECTION: Labeling of microtubules
20.2. Wash the cells for 2 min with 0.1 Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4 °C. (2/5)

SECTION: Labeling of microtubules
20.3. Wash the cells for 2 min with 0.1 Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4 °C. (3/5)

SECTION: Labeling of microtubules
20.4. Wash the cells for 2 min with 0.1 Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4 °C. (4/5)

SECTION: Labeling of microtubules
20.5. Wash the cells for 2 min with 0.1 Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4 °C. (5/5)

SECTION: Labeling of microtubules
24. Infiltrate the cells with one-part Durcupan ACM epoxy resin (44610, Sigma-Aldrich) to one-part anhydrous ethanol (1:1) for 30 min.

SECTION: Labeling of microtubules
3.5. After fixation, first wash the cells with CSB for  1 min  at  4 °C (5/5)
SECTION: Labeling of microtubules
3.4.     After fixation, first wash the cells with CSB for  1 min  at  4 °C (4/5)
SECTION: Labeling of microtubules
3.3. After fixation, first wash the cells with CSB for  1 min  at  4 °C (3/5)
SECTION: Labeling of microtubules
3.2. After fixation, first wash the cells with CSB for  1 min  at  4 °C (2/5)
SECTION: Labeling of microtubules
3.1. After fixation, first wash the cells with CSB for  1 min  at  4 °C (1/5)
SECTION: Labeling of microtubules
4. Treat with 0.1% saponin and 0.05% glycine in CSB for 20 mins at 4 °C while on a rocker.
SECTION: Labeling of microtubules
18. Wash the cells with 0.1 M sodium cacodylate buffer pH 7.4 (3 x 2 min) on ice.
SECTION: Labeling of microtubules
25. Infiltrate the cells 3 times with 100% Durcupan resin for 60 min each. 
SECTION: Labeling of microtubules
26. Do a final change of Durcupan resin and immediately place cells in a vacuum oven at 60 °C for 2880 min to harden.