Image Scanning Microscopy to Investigate Polycomb Protein Colocalization onto Chromatin
Abstract
:1. Introduction
2. Materials and Methods
2.1. Primary Antibodies
- Mouse anti-human EZH2 monoclonal antibody (3147S, Cell Signaling Technologies): 1/100 dilution.
- Rabbit anti-human BMI1 monoclonal antibody (6964S, Cell Signaling Technologies): 1/600 dilution.
- Rabbit anti-human RING1b monoclonal antibody (5694S, Cell Signaling Technologies): 1/250 dilution.
- Rabbit anti-human H3K27me3 monoclonal antibody (ab192985, AbCam): 1/1000 dilution.
2.2. Secondary Antibodies
- Donkey anti-mouse IgG AlexaFluor 488 (A32766, Life Technologies): 1/400 dilution.
- Donkey anti-rabbit IgG AlexaFluor 647 (A32795, Life Technologies): 1/400 dilution.
- Goat anti-rabbit IgG Abberior StarRed (41699, Merck Italia): 1/400 dilution.
2.3. Cell Culture
2.4. Cell Indirect Immunofluorescence Protocol
2.5. Confocal Fluorescence Microscopy
- Blurring (Gaussian, 1 pixel standard deviation) of the blue channel image.
- Background subtraction routine of Fiji (rolling ball radius: 50 pixels) of the blue channel image.
- Thresholding [22], filling holes and watershed separation of the blue channel image to yield an 8-bit mask of single nuclei.
- Particle analysis routine of Fiji of the green and red channels by using the mask of single nuclei.
2.6. Image Scanning Microscopy Setup
2.7. Image Scanning Microscopy Data Analysis
2.8. Determination of Image Resolution
2.9. Colocalization Analysis
2.10. Statistical Data Analysis and Fitting
3. Results and Discussion
3.1. Immunolabeling and Confocal Imaging of PcG Proteins and H3K27me3
3.2. Spatial Resolution Improvement by Image Scanning Microscopy
3.3. Colocalization by ISM
3.4. Single-cell Analysis of EZH2/H3K27me3 Correlation
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Nepita, I.; Piazza, S.; Ruglioni, M.; Cristiani, S.; Bosurgi, E.; Salvadori, T.; Vicidomini, G.; Diaspro, A.; Castello, M.; Bianchini, P.; et al. Image Scanning Microscopy to Investigate Polycomb Protein Colocalization onto Chromatin. Appl. Sci. 2023, 13, 1556. https://doi.org/10.3390/app13031556
Nepita I, Piazza S, Ruglioni M, Cristiani S, Bosurgi E, Salvadori T, Vicidomini G, Diaspro A, Castello M, Bianchini P, et al. Image Scanning Microscopy to Investigate Polycomb Protein Colocalization onto Chromatin. Applied Sciences. 2023; 13(3):1556. https://doi.org/10.3390/app13031556
Chicago/Turabian StyleNepita, Irene, Simonluca Piazza, Martina Ruglioni, Sofia Cristiani, Emanuele Bosurgi, Tiziano Salvadori, Giuseppe Vicidomini, Alberto Diaspro, Marco Castello, Paolo Bianchini, and et al. 2023. "Image Scanning Microscopy to Investigate Polycomb Protein Colocalization onto Chromatin" Applied Sciences 13, no. 3: 1556. https://doi.org/10.3390/app13031556
APA StyleNepita, I., Piazza, S., Ruglioni, M., Cristiani, S., Bosurgi, E., Salvadori, T., Vicidomini, G., Diaspro, A., Castello, M., Bianchini, P., Storti, B., & Bizzarri, R. (2023). Image Scanning Microscopy to Investigate Polycomb Protein Colocalization onto Chromatin. Applied Sciences, 13(3), 1556. https://doi.org/10.3390/app13031556