Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Formalin Fixation
2.3. High Pressure Freezing
2.4. Freeze Substitution and Rehydration
2.5. Pre-Embedding Immunogold Labeling and Processing for TEM
2.6. Immunofluorescence Labeling and Confocal Imaging
2.7. Control Experiments
3. Results
3.1. Antigen Presentation after Formalin Fixation of THP-1 Cells
3.2. A Combined HPF, FS, Rehydration, and Pre-Embedding Immunogold Method for Preserved Ultrastructure and Antigen Localization in the GCX of THP-1 Cells by TEM
3.3. Adjustment of the Method for Confocal Laser Scanning Microscopy
3.4. Control Experiments
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Antibody | Supplier | Host | Dilution |
---|---|---|---|
CD44 (IM7) FITC | Invitrogen 14-0441-85 | rat/IgG2b monoclonal | CLSM, 1:500 |
CD44 (IM7) | Invitrogen 14-0441-82 | rat/IgG2b monoclonal | TEM, 1:100 CLSM, 1:500 |
CS-56 | Abcam ab11570 | mouse/IgM monoclonal | TEM, 1:100 CLSM, 1:500 |
THP-1 Cells no. | Standard FS (+ OsO4) | Optimized FS (ø OsO4) | ||
---|---|---|---|---|
GCX (µm) | CB (µm) | GCX (µm) | CB (µm) | |
37 | 6.60 ± 0.30 * | 13.71 ± 0.32 ** | 6.45 ± 0.26 * | 13.11 ± 0.25 ** |
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Twamley, S.G.; Stach, A.; Heilmann, H.; Söhl-Kielczynski, B.; Stangl, V.; Ludwig, A.; Münster-Wandowski, A. Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx. Biology 2021, 10, 402. https://doi.org/10.3390/biology10050402
Twamley SG, Stach A, Heilmann H, Söhl-Kielczynski B, Stangl V, Ludwig A, Münster-Wandowski A. Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx. Biology. 2021; 10(5):402. https://doi.org/10.3390/biology10050402
Chicago/Turabian StyleTwamley, Shailey Gale, Anke Stach, Heike Heilmann, Berit Söhl-Kielczynski, Verena Stangl, Antje Ludwig, and Agnieszka Münster-Wandowski. 2021. "Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx" Biology 10, no. 5: 402. https://doi.org/10.3390/biology10050402