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Exp Eye Res
2015 Jul 01;136:86-90. doi: 10.1016/j.exer.2015.05.014.
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Preparation of Xenopus laevis retinal cryosections for electron microscopy.
Tam BM
,
Yang LL
,
Bogėa TH
,
Ross B
,
Martens G
,
Moritz OL
.
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Transmission electron microscopy is the gold standard for examination of photoreceptor outer segment morphology and photoreceptor outer segment abnormalities in transgenic animal models of retinal disease. Small vertebrates such as zebrafish and Xenopus laevis tadpoles have been used to generate retinal disease models and to study outer segment processes such as protein trafficking, and their breeding capabilities facilitate experiments involving large numbers of animals and conditions. However, electron microscopy processing and analysis of these very small eyes can be challenging. Here we present a methodology that facilitates processing of X. laevis tadpoleeyes for electron microscopy by introducing an intermediate cryosectioning step. This method reproducibly provides a well-oriented tissue block that can be sectioned with minimal effort by a non-expert, and also allows retroactive analysis of samples collected on slides for light microscopy.
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26008144
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Fig. 1.
Methodology for embedding of cryosections. Cryosections collected on glass slides are processed through osmium, ethanols, and infiltrated with resin (A), and finally embedded in a cylinder of resin (B). The resin cylinder is separated from the slide (C), and the block face is trimmed and sectioned (D). D: Fixed in 4% paraformaldehyde (4 PA).
Fig. 2.
TEM of photoreceptors derived from plastic-embedded retinal cryosections. Rod outer segments (ROS) (A) and cone outer segments (COS) (B) are well preserved with plasma membrane (PM), disk rims (Rim), disk lamellae (L) and calycal processes (CP) well resolved. The more complex organization of the cone outer segment disks is readily apparent. In addition, other intracellular structures are well preserved, including features of the connecting cilium (CC) and basal body (BB) (C), Golgi (D), endoplasmic reticulum (ER) (E), mitochondria (M) (F), and synaptic ribbons (SR) (G). C, G fixed in 4% paraformaldehyde (4 PA), all other images 4% paraformaldehyde 1% glutaraldehyde (P/G).
Fig. 3.
Artifacts associated with TEM of plastic-embedded retinal cryosections. A: cone photoreceptor outer segments lack definition in the region typically occupied by glycogen granules (GG). OD = oil droplet. Right: outer segment disk lamellae (L) are less well defined in samples fixed without glutaraldehyde, although disk rims (Rim) and plasma membrane (PM) are typically well defined. Left: 4% paraformaldehyde 1% glutaraldehyde (P/G). Right: 4% paraformaldehyde (4 PA).
