Polyclonal LC3B Antibodies Generate Non-Specific Staining in the Nucleus of Herpes Simplex Virus Type 1-Infected Cells: Caution in the Interpretation of LC3 Staining in the Immunofluorescence Analysis of Viral Infections
Abstract
1. Introduction
2. Results
2.1. LC3B Staining Is Observed in the Replication Compartments of HSV-1-Infected HOG Cells by Immunofluorescence
2.2. LC3B Staining Is Detected by Immunofluorescence in the RCs of HSV-1-Infected OPCs
2.3. Nuclear LC3B Staining During HSV-1 Infection Is Not Restricted to Oligodendroglial Cells
2.4. Nuclear LC3B Staining of HSV-1-Infected Cells Is Only Detected with Polyclonal Antibodies
2.5. The Isoforms LC3A and LC3C Are Not Detected in the Nucleus of Infected HOG Cells and OPCs
2.6. LC3B Is Not Detected in the Nuclear Fraction of HSV-1-Infected Cells by Immunoblotting
2.7. The Nuclear Staining Observed with Polyclonal LC3B Antibodies in HSV-1-Infected Cells Appears to Be Non-Specific
2.8. The Nuclear Staining Detected with Polyclonal LC3B Antibodies in HSV-1-Infected Cells Is Dependent on Viral Replication
2.9. Polyclonal LC3B Antibodies Are Appropriate for the Immunofluorescence Analysis of Autophagy in Non-Infected Cells
3. Discussion
4. Materials and Methods
4.1. Cell Lines
4.2. Viruses
4.3. Isolation and Culture of Oligodendrocyte Precursor Cells (OPCs)
4.4. Generation of the LC3B Knockout HOG Cell Line
4.5. Antibodies
4.6. Immunofluorescence Microscopy
4.7. Image Acquisition and Analysis
4.8. Immunoblot Analysis
4.9. Transient Transfection of the GFP-LC3 Plasmid
4.10. Inhibition of HSV-1 Genome Replication
4.11. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
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Ripa, I.; Andreu, S.; Galdo, D.; Caballero, O.; Bello-Morales, R.; López-Guerrero, J.A. Polyclonal LC3B Antibodies Generate Non-Specific Staining in the Nucleus of Herpes Simplex Virus Type 1-Infected Cells: Caution in the Interpretation of LC3 Staining in the Immunofluorescence Analysis of Viral Infections. Int. J. Mol. Sci. 2025, 26, 6682. https://doi.org/10.3390/ijms26146682
Ripa I, Andreu S, Galdo D, Caballero O, Bello-Morales R, López-Guerrero JA. Polyclonal LC3B Antibodies Generate Non-Specific Staining in the Nucleus of Herpes Simplex Virus Type 1-Infected Cells: Caution in the Interpretation of LC3 Staining in the Immunofluorescence Analysis of Viral Infections. International Journal of Molecular Sciences. 2025; 26(14):6682. https://doi.org/10.3390/ijms26146682
Chicago/Turabian StyleRipa, Inés, Sabina Andreu, Daniel Galdo, Oliver Caballero, Raquel Bello-Morales, and José Antonio López-Guerrero. 2025. "Polyclonal LC3B Antibodies Generate Non-Specific Staining in the Nucleus of Herpes Simplex Virus Type 1-Infected Cells: Caution in the Interpretation of LC3 Staining in the Immunofluorescence Analysis of Viral Infections" International Journal of Molecular Sciences 26, no. 14: 6682. https://doi.org/10.3390/ijms26146682
APA StyleRipa, I., Andreu, S., Galdo, D., Caballero, O., Bello-Morales, R., & López-Guerrero, J. A. (2025). Polyclonal LC3B Antibodies Generate Non-Specific Staining in the Nucleus of Herpes Simplex Virus Type 1-Infected Cells: Caution in the Interpretation of LC3 Staining in the Immunofluorescence Analysis of Viral Infections. International Journal of Molecular Sciences, 26(14), 6682. https://doi.org/10.3390/ijms26146682