Abstract
Detection of the protein PCNA (proliferating cell nuclear antigen) is used to identify cells in the S phase of the cell cycle to indicate cellular proliferation. Here we describe our method to detect PCNA expression by microglia and macrophages in retinal cryosections. We have used this procedure with zebrafish tissue, but this procedure could be applied to cryosections from any organism. Retinal cryosections are subjected to a heat-mediated antigen retrieval step in Citrate Buffer, then immunostained with antibodies to label PCNA and microglia/macrophages, and counterstained for cell nuclei. After fluorescent microscopy, the number of total and PCNA+ microglia/macrophages can be quantified and normalized to compare across samples and groups.
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References
Mitchell DM, Lovel AG, Stenkamp DL (2018) Dynamic changes in microglial and macrophage characteristics during degeneration and regeneration of the zebrafish retina. J Neuroinflamm 15:163. https://doi.org/10.1186/s12974-018-1185-6
Mitchell DM, Sun C, Hunter SS, New DD, Stenkamp DL (2019) Regeneration associated transcriptional signature of retinal microglia and macrophages. Sci Rep-uk 9:4768. https://doi.org/10.1038/s41598-019-41298-8
Blume ZI, Lambert JM, Lovel AG, Mitchell DM (2020) Microglia in the developing retina couple phagocytosis with the progression of apoptosis via P2RY12 signaling. Dev Dynam 249:723–740. https://doi.org/10.1002/dvdy.163
Conedera FM, Pousa AMQ, Mercader N, Tschopp M, Enzmann V (2019) Retinal microglia signaling affects Müller cell behavior in the zebrafish following laser injury induction. Glia 67:1150–1166. https://doi.org/10.1002/glia.23601
Walsh CE, Hitchcock PF (2017) Progranulin regulates neurogenesis in the developing vertebrate retina. Dev Neurobiol 77:1114–1129. https://doi.org/10.1002/dneu.22499
Rovira M, Miserocchi M, Montanari A, Hammou L, Chomette L, Pozo J, Imbault V, Bisteau X, Wittamer V (2022) Zebrafish Galectin 3 binding protein is the target antigen of the microglial 4C4 monoclonal antibody. Dev Dyn. https://doi.org/10.1002/dvdy.549
Craig SEL, Calinescu A-A, Hitchcock PF (2008) Identification of the molecular signatures integral to regenerating photoreceptors in the retina of the zebra fish. J Ocular Biology Dis Informatics 1:73. https://doi.org/10.1007/s12177-008-9011-5
Bernardos RL, Barthel LK, Meyers JR, Raymond PA (2007) Late-stage neuronal progenitors in the retina are radial Müller glia that function as retinal stem cells. J Neurosci 27:7028–7040. https://doi.org/10.1523/jneurosci.1624-07.2007
Silva NJ, Nagashima M, Li J, Kakuk-Atkins L, Ashrafzadeh M, Hyde DR, Hitchcock PF (2020) Inflammation and matrix metalloproteinase 9 (Mmp-9) regulate photoreceptor regeneration in adult zebrafish. Glia 68:1445–1465. https://doi.org/10.1002/glia.23792
Raymond PA, Barthel LK, Bernardos RL, Perkowski JJ (2006) Molecular characterization of retinal stem cells and their niches in adult zebrafish. BMC Dev Biol 6:36. https://doi.org/10.1186/1471-213x-6-36
Ellett F, Pase L, Hayman JW, Andrianopoulos A, Lieschke GJ (2011) mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood 117:e49–e56. https://doi.org/10.1182/blood-2010-10-314120
White DT, Sengupta S, Saxena MT, Xu Q, Hanes J, Ding D, Ji H, Mumm JS (2017) Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina. Proc National Acad Sci 114:E3719–E3728. https://doi.org/10.1073/pnas.1617721114
Acknowledgments
This work was supported by funding from Idaho INBRE NIH Grant P20 GM103408 and NIH Grant R01EY030467 to DMM. We thank Dr. Deborah Stenkamp for review of previous versions of the chapter.
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Lovel, A.G., Mitchell, D.M. (2023). PCNA Staining of Retinal Cryosections to Assess Microglial/Macrophage Proliferation. In: Udvadia, A.J., Antczak, J.B. (eds) Axon Regeneration. Methods in Molecular Biology, vol 2636. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3012-9_21
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DOI: https://doi.org/10.1007/978-1-0716-3012-9_21
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