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Abstract
Identifying endogenous tissue stem cells remains a key challenge in developmental and regenerative biology. To distinguish and molecularly characterise stem cell populations in large heterogeneous tissues, the combination of cytochemical cell markers with ultrastructural morphology is highly beneficial. Here, we realise this through workflows of multi-resolution immuno-correlative light and electron microscopy (iCLEM) methodologies. Taking advantage of the antigenicity preservation of the Tokuyasu technique, we have established robust protocols and workflows and provide a side-by-side comparison of iCLEM used in combination with scanning EM (SEM), scanning TEM (STEM), or transmission EM (TEM). Evaluation of the applications and advantages of each method highlights their practicality for the identification, quantification, and characterization of heterogeneous cell populations in small organisms, organs, or tissues in healthy and diseased states. The iCLEM techniques are broadly applicable and can use either genetically encoded or cytochemical markers on plant, animal and human tissues. We demonstrate how these protocols are particularly suited for investigating neural stem and progenitor cell populations of the vertebrate nervous system.
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Details
1 Ramaciotti Centre for Cryo EM, Monash University, Melbourne, Australia (GRID:grid.1002.3) (ISNI:0000 0004 1936 7857); Electron Microscopy Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany (GRID:grid.4709.a) (ISNI:0000 0004 0495 846X)
2 Monash University, Australian Regenerative Medicine Institute, Melbourne, Australia (GRID:grid.1002.3) (ISNI:0000 0004 1936 7857); University of Manitoba, Department of Human Anatomy and Cell Science, Rady Faculty of Health Sciences, Winnipeg, Canada (GRID:grid.21613.37) (ISNI:0000 0004 1936 9609)
3 Monash University, Australian Regenerative Medicine Institute, Melbourne, Australia (GRID:grid.1002.3) (ISNI:0000 0004 1936 7857)
4 Ramaciotti Centre for Cryo EM, Monash University, Melbourne, Australia (GRID:grid.1002.3) (ISNI:0000 0004 1936 7857); Monash University, Department of Biochemistry, Biomedicine Discovery Institute, Melbourne, Australia (GRID:grid.1002.3) (ISNI:0000 0004 1936 7857)