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Monoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133)
2016, Thamm, Kristina, Graupner, Sylvi, Werner, Carsten, Huttner, Wieland B., Corbeil, Denis, Nabi, Ivan R
The pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.
Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength
2016, Lu-Walther, Hui-Wen, Hou, Wenya, Kielhorn, Martin, Arai, Yoshiyuki, Nagai, Takeharu, Kessels, Michael M., Qualmann, Britta, Heintzmann, Rainer
Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.
Scanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying
2021, Schu, Moritz, Terriac, Emmanuel, Koch, Marcus, Paschke, Stephan, Lautenschläger, Franziska, Flormann, Daniel A.D.
The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.
Accurate in vivo tumor detection using plasmonic-enhanced shifted-excitation Raman difference spectroscopy (SERDS)
2021, Strobbia, Pietro, Cupil-Garcia, Vanessa, Crawford, Bridget M., Fales, Andrew M., Pfefer, T. Joshua, Liu, Yang, Maiwald, Martin, Sumpf, Bernd, Vo-Dinh, Tuan
For the majority of cancer patients, surgery is the primary method of treatment. In these cases, accurately removing the entire tumor without harming surrounding tissue is critical; however, due to the lack of intraoperative imaging techniques, surgeons rely on visual and physical inspection to identify tumors. Surface-enhanced Raman scattering (SERS) is emerging as a non-invasive optical alternative for intraoperative tumor identification, with high accuracy and stability. However, Raman detection requires dark rooms to work, which is not consistent with surgical settings. Methods: Herein, we used SERS nanoprobes combined with shifted-excitation Raman difference spectroscopy (SERDS) detection, to accurately detect tumors in xenograft murine model. Results: We demonstrate for the first time the use of SERDS for in vivo tumor detection in a murine model under ambient light conditions. We compare traditional Raman detection with SERDS, showing that our method can improve sensitivity and accuracy for this task. Conclusion: Our results show that this method can be used to improve the accuracy and robustness of in vivo Raman/SERS biomedical application, aiding the process of clinical translation of these technologies. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Simultaneous statistical inference for epigenetic data
2015, Schildknecht, Konstantin, Olek, Sven, Dickhaus, Thorsten
Epigenetic research leads to complex data structures. Since parametric model assumptions for the distribution of epigenetic data are hard to verify we introduce in the present work a nonparametric statistical framework for two-group comparisons. Furthermore, epigenetic analyses are often performed at various genetic loci simultaneously. Hence, in order to be able to draw valid conclusions for specific loci, an appropriate multiple testing correction is necessary. Finally, with technologies available for the simultaneous assessment of many interrelated biological parameters (such as gene arrays), statistical approaches also need to deal with a possibly unknown dependency structure in the data. Our statistical approach to the nonparametric comparison of two samples with independent multivariate observables is based on recently developed multivariate multiple permutation tests. We adapt their theory in order to cope with families of hypotheses regarding relative effects. Our results indicate that the multivariate multiple permutation test keeps the pre-assigned type I error level for the global null hypothesis. In combination with the closure principle, the family-wise error rate for the simultaneous test of the corresponding locus/parameter-specific null hypotheses can be controlled. In applications we demonstrate that group differences in epigenetic data can be detected reliably with our methodology.