PUBLICATION

Immuno correlative light and electron microscopy on tokuyasu cryosections

Authors
Oorschot, V.M., Sztal, T.E., Bryson-Richardson, R.J., Ramm, G.
ID
ZDB-PUB-141008-8
Date
2014
Source
Methods in cell biology   124: 241-58 (Chapter)
Registered Authors
Bryson-Richardson, Robert
Keywords
CLEM, GFP, Immuno-EM, Tokuyasu, Zebrafish
MeSH Terms
  • Animals
  • Cryopreservation
  • Fiducial Markers
  • Frozen Sections
  • Green Fluorescent Proteins/biosynthesis
  • Image Processing, Computer-Assisted
  • Microscopy, Electron, Transmission/methods
  • Microscopy, Fluorescence/methods
  • Microscopy, Immunoelectron/methods
  • Muscle, Skeletal/metabolism
  • Muscle, Skeletal/ultrastructure*
  • Tissue Embedding
  • Tissue Fixation
  • Zebrafish
PubMed
25287844 Full text @ Meth. Cell. Biol.
Abstract
Finding a rare structure by electron microscopy is the equivalent of finding a "needle in a haystack." Correlative light- and immunoelectron microscopy (CLEM) on Tokuyasu cryosections is a sophisticated technique to address this challenge. Hereby, fluorescently labeled structures of interest are identified in an overview image by light microscopy and subsequently traced in electron microscopy. While the direct transfer and imaging of the same sections from optical to electron microscopy enables straightforward correlation, the sample preparation is crucial and technically demanding. We provide a detailed guide outlining the critical steps for sample embedding, cryosectioning, immunolabeling, and imaging. In the example provided, we use CLEM to trace aggregates formed in a zebrafish myopathy model expressing enhanced green fluorescent protein (eGFP) tagged actin. In our case, only a few muscle fibers express eGFP-actin with a subset of fibers containing aggregates. By fluorescence microscopy, we are able to identify the aggregates in the zebrafish tissue, and we subsequently, use immunoelectron microscopy to image the same structures at high resolution. The CLEM method described here using Tokuyasu cryosections can be applied to a large range of samples including small organisms, tissue samples, and cells.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping