During erythropoiesis, erythroblasts undergo dramatic morphological changes to produce mature erythrocytes. Many unanswered questions regarding the molecular mechanisms behind these changes can be addressed with high-resolution fluorescence imaging. Immunofluoresence staining enables localization of specific molecules, organelles, and membrane components in intact cells at different phases of erythropoiesis. Confocal laser scanning microscopy can provide high-resolution, three-dimensional images of stained structures, which can be used to dissect the molecular mechanisms driving erythropoiesis. The sample preparation, staining procedure, imaging parameters, and image analysis methods used directly affect the quality of the confocal images and the amount and accuracy of information that they can provide. Here, we describe methods to dissect erythropoietic tissues from mice, to perform immunofluorescence staining and confocal imaging of various molecules, organelles and structures of interest in erythroblasts, and to present and quantitatively analyze the data obtained in these fluorescence images.
Erythroblast Fetal liver Bone marrow Spleen Immunofluorescence Confocal microscopy
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Wong P, Hattangadi SM, Cheng AW et al (2011) Gene induction and repression during terminal erythropoiesis are mediated by distinct epigenetic changes. Blood 118:e128–e138CrossRefPubMedPubMedCentralGoogle Scholar
Koury ST, Koury MJ, Bondurant MC (1989) Cytoskeletal distribution and function during the maturation and enucleation of mammalian erythroblasts. J Cell Biol 109:3005–3013CrossRefPubMedGoogle Scholar
Pawley JB (ed) (2006) Handbook of biological confocal microscopy. Springer, New York, NYGoogle Scholar
Nowak RB, Papoin J, Gokhin DS, Casu C, Rivella S, Lipton JM, Blanc L, Fowler VM (2017) Tropomodulin 1 controls erythroblast enucleation via regulation of F-actin in the enucleosome. Blood 130(9):1144–1155CrossRefPubMedGoogle Scholar
Sui Z, Nowak RB, Bacconi A, Kim NE, Liu H, Li J, Wickrema A, An X, Fowler VM (2014) Tropomodulin3-null mice are embryonic lethal with anemia due to impaired erythroid terminal differentiation in the fetal liver. Blood 123:758–767CrossRefPubMedPubMedCentralGoogle Scholar
Fischer AH, Jacobson KA, Rose J, Zeller R (2008) Preparation of slides and coverslips for microscopy. Cold Spring Harb Protoc 2008:pdb.prot4988–pdb.prot4988Google Scholar
Waterman-Storer CM (2001) Microtubule/organelle motility assays. Curr Protoc Cell Biol. Chapter 13, Unit 13.1Google Scholar
Costes SV, Daelemans D, Cho EH, Dobbin Z, Pavlakis G, Lockett S (2004) Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys J 86:3993–4003CrossRefPubMedPubMedCentralGoogle Scholar
Pantel K, Loeffler M, Bungart B, Wichmann HE (1990) A mathematical model of erythropoiesis in mice and rats. Part 4: Differences between bone marrow and spleen. Cell Tissue Kinet 23:283–297PubMedGoogle Scholar
Latunde-Dada GO, McKie AT, Simpson RJ (2006) Animal models with enhanced erythropoiesis and iron absorption. Biochim Biophys Acta 1762:414–423CrossRefPubMedGoogle Scholar
Bernas T, ZarÉBski M, Cook RR, Dobrucki JW (2004) Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux. J Microsc 215:281–296CrossRefPubMedGoogle Scholar