May 24, 2024
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
- Mason Mackey1,2,
- Mark H. Ellisman1,2,
- Stephen Adams1,2
- 1UC San Diego Health Sciences;
- 2National Center for Microscopy and Imaging Research
External link: https://www.biorxiv.org/content/10.1101/2023.08.25.554352v2
Protocol Citation: Mason Mackey, Mark H. Ellisman, Stephen Adams 2024. 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. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5dpdg47/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 17, 2024
Last Modified: May 24, 2024
Protocol Integer ID: 100230
Keywords: TEM, DAB oxidation, oxidation reaction, Correlated Light and Electron Microscopy, Fe-TAML, small molecule peroxidase
Funders Acknowledgement:
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)
Abstract
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.
Materials
CSB buffer:
| A | B | |
| Pipes buffer | 10 mM | |
| NaCl | 150 mM | |
| EGTA | 5 mM | |
| MgCl2 | 5 mM | |
| Glucose monohydrate, pH 6.8 | 5 mM |
- Anti-β-Tubulin antibody, Mouse monoclonal, clone TUB 2.1Merck MilliporeSigma (Sigma-Aldrich)Catalog #T5201
- 3,3′-DiaminobenzidineMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8001- 10G
- Durcupan™ ACMMerck MilliporeSigma (Sigma-Aldrich)Catalog #44610
- Fe-TAML-peg4-Cy5-goat anti-mouse IgG
- BSA Sigma Catalog #A8022-100G
Labeling of microtubules
Labeling of microtubules
2d 17h 59m
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 00:05:00 and for 00:25:00 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 |
30m
After fixation, first wash the cells with CSB (5 x 1 min) at 4 °C .
After fixation, first wash the cells with CSB for 00:01:00 at 4 °C (1/5)
1m
After fixation, first wash the cells with CSB for 00:01:00 at 4 °C (2/5)
1m
After fixation, first wash the cells with CSB for 00:01:00 at 4 °C (3/5)
1m
After fixation, first wash the cells with CSB for 00:01:00 at 4 °C (4/5)
1m
After fixation, first wash the cells with CSB for 00:01:00 at 4 °C (5/5)
1m
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 00:01:00 at 4 °C . (1/3)
1m
Wash the cells in CSB buffer with 0.05% glycine 00:01:00 at 4 °C . (2/3)
1m
Wash the cells in CSB buffer with 0.05% glycine 00:01:00 at 4 °C . (3/3)
1m
Block the cells with 1% BSA (A8022-100G, Sigma), 1% normal goat serum (NGS) and 0.05% glycine in CSB for 00:20:00 at 4 °C .
20m
Incubate the cells with primary mouse monoclonal antibody to β-tubulin (300-fold dilution, clone Tub2.1, T5201, Sigma) for 03:00:00 at 4 °C in 1% BSA, 1% NGS and 0.05% glycine in CSB buffer.
3h
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 00:03:00 at 4 °C . (1/5)
3m
Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 00:03:00 at 4 °C . (2/5)
3m
Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 00:03:00 at 4 °C . (3/5)
3m
Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 00:03:00 at 4 °C . (4/5)
3m
Wash with 1% BSA, 1% NGS and 0.05% glycine in CSB 00:03:00 at 4 °C . (5/5)
3m
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 Overnight at 4 °C . [Adams et al., 2023].
8h
Then wash the cells (5 x 1 min) with CSB at 4°C.
Wash the cells for 00:01:00 with CSB at 4 °C . (1/5)
1m
Wash the cells for 00:01:00 with CSB at 4 °C . (2/5)
1m
Wash the cells for 00:01:00 with CSB at 4 °C . (3/5)
1m
Wash the cells for 00:01:00 with CSB at 4 °C . (4/5)
1m
Wash the cells for 00:01:00 with CSB at 4 °C . (5/5)
1m
Fix the cells with 2% glutaraldehyde in CSB for 00:20:00 at 4 °C .
20m
Wash the cells (5 x 1 min) with CSB at 4°C.
Wash the cells for 00:01:00 with CSB at 4 °C . (1/5)
1m
Wash the cells for 00:01:00 with CSB at 4 °C . (2/5)
1m
Wash the cells for 00:01:00 with CSB at 4 °C . (3/5)
1m
Wash the cells for 00:01:00 with CSB at 4 °C . (4/5)
1m
Wash the cells for 00:01:00 with CSB at 4 °C . (5/5)
1m
Image the cells for fluorescence labeling.
Dissolve and add 5.4 mg of 3,3’- Diaminobenzidine (DAB) (D8001-10G, Sigma-Aldrich) in 1.0 mL of 0.1 Mass Percent 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.
Wash the cells (2 x 2 min) with 50 mM Bicine 100 mM NaCl pH 8.3 at 4°C.
Wash the cells for 00:02:00 with 50 millimolar (mM) Bicine 100 millimolar (mM) NaCl pH 8.3 at 4 °C . (1/2)
2m
Wash the cells for 00:02:00 with 50 millimolar (mM) Bicine 100 millimolar (mM) NaCl pH 8.3 at 4 °C . (2/2)
2m
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 01:00:00 -01:30:00 .
1h 30m
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 50 millimolar (mM) Bicine 100 millimolar (mM) NaCl pH 8.3 for 00:02:00 On ice . (1/2)
2m
Wash the cells with 50 millimolar (mM) Bicine 100 millimolar (mM) NaCl pH 8.3 for 00:02:00 On ice . (2/2)
2m
Wash the cells with 0.1 M sodium cacodylate buffer pH 7.4 (3 x 2 min) on ice.
Wash the cells with 0.1 Molarity (M) sodium cacodylate buffer pH 7.4 for 00:02:00 . (1/3)
2m
Wash the cells with 0.1 Molarity (M) sodium cacodylate buffer pH 7.4 for 00:02:00 . (2/3)
2m
Wash the cells with 0.1 Molarity (M) sodium cacodylate buffer pH 7.4 for 00:02:00 . (3/3)
2m
Do a final primary fixation with 2% glutaraldehyde in 2 millimolar (mM) CaCl2 0.1 Molarity (M) sodium cacodylate pH 7.4 for 00:30:00 at 4 °C .
30m
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 00:02:00 with 0.1 Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4 °C . (1/5)
2m
Wash the cells for 00:02:00 with 0.1 Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4 °C . (2/5)
2m
Wash the cells for 00:02:00 with 0.1 Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4 °C . (3/5)
2m
Wash the cells for 00:02:00 with 0.1 Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4 °C . (4/5)
2m
Wash the cells for 00:02:00 with 0.1 Molarity (M) sodium cacodylate (18851, Ted Pella) pH 7.4 at 4 °C . (5/5)
2m
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 00:30:00 at 4 °C .
30m
Wash the cells (5 x 2 min) with ddH2O at 4°C.
Wash the cells for 00:02:00 with ddH2O at 4 °C . (1/5)
2m
Wash the cells for 00:02:00 with ddH2O at 4 °C . (2/5)
2m
Wash the cells for 00:02:00 with ddH2O at 4 °C . (3/5)
2m
Wash the cells for 00:02:00 with ddH2O at 4 °C . (4/5)
2m
Wash the cells for 00:02:00 with ddH2O at 4 °C . (5/5)
2m
Dehydrate the cells by 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 the cells with one-part Durcupan ACM epoxy resin (44610, Sigma-Aldrich) to one-part anhydrous ethanol (1:1) for 00:30:00 .
30m
Infiltrate the cells 3 times with 100% Durcupan resin for 01:00:00 each.
1h
Do a final change of Durcupan resin and immediately place cells in a vacuum oven at 60 °C for 48:00:00 to harden.
2d
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.