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

Culture HEK293T cells on MatTek plates containing poly-D-lysine coated glass bottom No.0 coverslips in DMEM supplement with 10% fetal bovine serum.

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 0h 5m 0s and for 0h 25m 0s at 4°C.

CSB buffer:

A	B
Pipes buffer	10 mM
NaCl	150 mM
EGTA	5 mM
MgCl2	5 mM
Glucose monohydrate, pH 6.8	5 mM

After fixation, first wash the cells with CSB (5 x 1 min) at 4°C.

After fixation, first wash the cells with CSB for 0h 1m 0s at 4°C (1/5)

After fixation, first wash the cells with CSB for 0h 1m 0s at 4°C (2/5)

After fixation, first wash the cells with CSB for 0h 1m 0s at 4°C (3/5)

After fixation, first wash the cells with CSB for  `0h 1m 0s`  at  `4°C` (4/5)

After fixation, first wash the cells with CSB for 0h 1m 0s at 4°C (5/5)

Treat with 0.1% saponin and 0.05% glycine in CSB for 20 mins at 4°C while on a rocker.

Wash the cells in CSB buffer with 0.05% glycine (3 x 1 min) at 4°C.

Wash the cells in CSB buffer with 0.05% glycine 0h 1m 0s at 4°C. (1/3)

Wash the cells in CSB buffer with 0.05% glycine 0h 1m 0s at 4°C. (2/3)

Wash the cells in CSB buffer with 0.05% glycine 0h 1m 0s at 4°C. (3/3)

Block the cells with 1% BSA (A8022-100G, Sigma), 1% normal goat serum (NGS) and 0.05% glycine in CSB for 0h 20m 0s at 4°C.

Incubate the cells with primary mouse monoclonal antibody to β-tubulin (300-fold dilution, clone Tub2.1, T5201, Sigma) for 3h 0m 0s at 4°C in 1% BSA, 1% NGS and 0.05% glycine in CSB buffer.

Remove primary antibody and wash with 1% BSA, 1% NGS and 0.05% glycine in CSB (5 x 3 min) at 4°C.

Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 0h 3m 0s at 4°C. (1/5)

Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 0h 3m 0s at 4°C. (2/5)

Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 0h 3m 0s at 4°C. (3/5)

Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 0h 3m 0s at 4°C. (4/5)

Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 0h 3m 0s at 4°C. (5/5)

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 0h 3m 0s at 4°C. [Adams et al., 2023].

Then wash the cells (5 x 1 min) with CSB at 4°C.

Wash the cells for 0h 1m 0s with CSB at 4°C. (1/5)

Wash the cells for 0h 1m 0s with CSB at 4°C. (2/5)

Wash the cells for 0h 1m 0s with CSB at 4°C. (3/5)

Wash the cells for 0h 1m 0s with CSB at 4°C. (4/5)

Wash the cells for 0h 1m 0s with CSB at 4°C. (5/5)

Fix the cells with 2% glutaraldehyde in CSB for 0h 20m 0s at 4°C.

Wash the cells (5 x 1 min) with CSB at 4°C.

Wash the cells for 0h 1m 0s with CSB at 4°C. (1/5)

Wash the cells for 0h 1m 0s with CSB at 4°C. (2/5)

Wash the cells for 0h 1m 0s with CSB at 4°C. (3/5)

Wash the cells for 0h 1m 0s with CSB at 4°C. (4/5)

Wash the cells for 0h 1m 0s with CSB at 4°C. (5/5)

Image the cells for fluorescence labeling.

Dissolve and add 5.4mg of 3,3’- Diaminobenzidine (DAB) (D8001-10G, Sigma-Aldrich) in 1.0mL of 0.1 HCl and 9.0mL of 50millimolar (mM) Bicine 100millimolar (mM) NaCl pH 8.3 with 10µL H₂O₂ (final, 40millimolar (mM) from 30% stock) to the DAB solution.

Wash the cells (2 x 2 min) with 50 mM Bicine 100 mM NaCl pH 8.3 at 4°C.

Wash the cells for 0h 2m 0s with 50millimolar (mM) Bicine 100millimolar (mM)NaCl pH 8.3 at 4°C. (1/2)

Wash the cells for 0h 2m 0s with 50millimolar (mM) Bicine 100millimolar (mM)NaCl pH 8.3 at 4°C. (2/2)

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 1h 0m 0s-1h 30m 0s.

Remove the DAB solution, and wash the cells with 50 mM Bicine 100mM NaCl pH 8.3 (2 x 2 min) on ice.

Wash the cells with 50millimolar (mM) Bicine 100millimolar (mM) NaCl pH 8.3 for 0h 2m 0s On ice . (1/2)

Wash the cells with 50millimolar (mM) Bicine 100millimolar (mM) NaCl pH 8.3 for 0h 2m 0s On ice . (2/2)

Wash the cells with 0.1 M sodium cacodylate buffer pH 7.4 (3 x 2 min) on ice.

Wash the cells with 0.1Molarity (M)sodium cacodylate buffer pH 7.4 for 0h 2m 0s. (1/3)

Wash the cells with 0.1Molarity (M)sodium cacodylate buffer pH 7.4 for 0h 2m 0s. (2/3)

Wash the cells with 0.1Molarity (M)sodium cacodylate buffer pH 7.4 for 0h 2m 0s. (3/3)

Do a final primary fixation with 2% glutaraldehyde in 2millimolar (mM) CaCl₂ 0.1Molarity (M) sodium cacodylate pH 7.4 for 0h 30m 0s at 4°C.

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.

Wash the cells for 0h 2m 0s with 0.1Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4°C. (1/5)

Wash the cells for 0h 2m 0s with 0.1Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4°C. (2/5)

Wash the cells for 0h 2m 0s with 0.1Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4°C. (3/5)

Wash the cells for 0h 2m 0s with 0.1Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4°C. (4/5)

Wash the cells for 0h 2m 0s with 0.1Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4°C. (5/5)

All cells are post-fixed with 1% osmium tetroxide (19150, Electron Microscopy Sciences) containing 0.8% potassium ferrocyanide, 2millimolar (mM) CaCl₂ and in 0.1Molarity (M) sodium cacodylate pH 7.4 for 0h 30m 0s at 4°C.

Wash the cells (5 x 2 min) with ddH₂O at 4°C.

Wash the cells for 0h 2m 0s with ddH₂O at 4°C. (1/5)

Wash the cells for 0h 2m 0s with ddH₂O at 4°C. (2/5)

Wash the cells for 0h 2m 0s with ddH₂O at 4°C. (3/5)

Wash the cells for 0h 2m 0s with ddH₂O at 4°C. (4/5)

Wash the cells for 0h 2m 0s with ddH₂O at 4°C. (5/5)

Dehydrate the cells by 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 the cells with one-part Durcupan ACM epoxy resin (44610, Sigma-Aldrich) to one-part anhydrous ethanol (1:1) for 0h 30m 0s.

Infiltrate the cells 3 times with 100% Durcupan resin for 1h 0m 0s each.

Do a final change of Durcupan resin and immediately place cells in a vacuum oven at 60°C for 48h 0m 0s to harden.