Improved antigen retrieval in freeze-fracture cytochemistry by evaporation of carbon as first replication layer

Schlörmann, Wiebke; John, Markus; Steiniger, Frank; Westermann, Martin; Richter, Walter

doi:10.1007/s00418-007-0283-9

Cited by 14 publications

(11 citation statements)

References 46 publications

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“…We have noticed that labeling for ganglioside GM1 is drastically improved when the evaporation order is reversed, i.e., by evaporating C Wrst followed by Pt/C (Fujita et al 2007); a similar result was reported for some proteins (Masugi-Tokita et al 2007;Schlormann et al 2007). Why this improvement occurs by changing the order of evaporation has not been studied to date.…”

Section: Introductionsupporting

confidence: 60%

Quantitative retention of membrane lipids in the freeze-fracture replica

Fujita

Fujimoto

2007

Histochem Cell Biol

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SDS-digested freeze-fracture replicas have been used as a substrate for immunoelectron microscopy to determine localization of membrane proteins and lipids. We, as well as others, have noticed that replicas prepared by first evaporating carbon are labeled more efficiently than conventional preparations in which platinum/carbon is evaporated first followed by carbon. In the present study, we examined whether the superior labeling in the carbon-first replica is caused by better retention of membrane molecules during SDS digestion. We used phosphatidylcholine liposomes as a model sample and measured the amount of inorganic phosphorus retained in the SDS-digested replica. The result showed that there was equivalent retention of inorganic phosphate among replicas prepared in different ways, indicating that labeling intensity on the replica did not correlate with the retention ratio. Interestingly, despite a similar retention ratio, replicas made of carbon alone gave far less labeling for ganglioside GM1 and phosphatidylcholine than replicas prepared by carbon followed by platinum/carbon. These results suggest that probes can bind to lipids captured by carbon more efficiently than those captured by platinum. Nonetheless, evaporation of platinum after carbon is indispensable for proper labeling.

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Section: Introductionsupporting

confidence: 60%

Quantitative retention of membrane lipids in the freeze-fracture replica

Fujita

Fujimoto

2007

Histochem Cell Biol

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“…Frozen slices were knife-cleaved in either a JEOL/RMC JFD 9010C or JFD II freeze-fracture device equipped with a −170°C liquid-nitrogen-cooled specimen cryoshroud and a liquid-helium-cooled cryopump (Rash and Yasumura, 1999). Freeze-fractured samples were either coated with 1-5 nm of carbon before replication [to increase labeling efficiency (Fujimoto, 1995; Schlörmann et al, 2007) but also decreases image resolution], or were replicated with 1-2 nm of platinum immediately after fracture (Rash and Yasumura, 1999). All replicated samples were stabilized by coating with 10-20 nm of carbon; the samples were removed and placed into a liquid nitrogen-cooled-cooled chamber that maintained the atmosphere at ca.…”

Section: Methodsmentioning

confidence: 99%

Heterotypic gap junctions at glutamatergic mixed synapses are abundant in goldfish brain

et al. 2015

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Gap junctions provide for direct intercellular electrical and metabolic coupling. The abundance of gap junctions at “large myelinated club ending” synapses on Mauthner cells of the teleost brain provided a convenient model to correlate anatomical and physiological properties of electrical synapses. There, presynaptic action potentials were found to evoke short-latency electrical “pre-potentials” immediately preceding their accompanying glutamate-induced depolarizations, making these the first unambiguously identified “mixed” (i.e., chemical plus electrical) synapses in the vertebrate CNS. We recently showed that gap junctions at these synapses exhibit asymmetric electrical resistance (i.e., electrical rectification), which we correlated with total molecular asymmetry of connexin composition in their apposing gap junction hemiplaques, with Cx35 restricted to axon terminal hemiplaques and Cx34.7 restricted to apposing Mauthner cell plasma membranes. We now show that similarly heterotypic neuronal gap junctions are abundant throughout goldfish brain, with labeling exclusively for Cx35 in presynaptic hemiplaques and exclusively for Cx34.7 in postsynaptic hemiplaques. Moreover, the vast majority of these asymmetric gap junctions occur at glutamatergic axon terminals. The widespread distribution of heterotypic gap junctions at glutamatergic mixed synapses throughout goldfish brain and spinal cord implies that pre- vs. postsynaptic asymmetry at electrical synapses evolved early in the chordate lineage. We propose that the advantages of the molecular and functional asymmetry of connexins at electrical synapses that are so prominently expressed in the teleost CNS are unlikely to have been abandoned in higher vertebrates. However, to create asymmetric coupling in mammals, where most gap junctions are composed of Cx36 on both sides, would require some other mechanism, such as differential phosphorylation of connexins on opposite sides of the same gap junction or on asymmetric differences in the complement of their scaffolding and regulatory proteins.

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“…In recent investigations we established and enhanced the freeze-fracture immunolabeling technique according to Fujimoto (1997) in order to study the caveolin distribution at plasma membrane caveolae (Westermann et al 2005;Schloermann et al 2007). In the technique, sodium dodecyl sulfate (SDS) was used to clean freeze-fracture replicas obtained from chemically unWxed, rapidly frozen cells.…”

Section: Resultsmentioning

confidence: 99%

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation

Schlörmann

Steiniger

Richter

et al. 2009

Histochem Cell Biol

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Caveolae were defined as flask- or omega-shaped plasma membrane invaginations, abundant in adipocytes, fibroblasts, endothelial and smooth muscle cells. The major protein component of caveolar membranes is an integral membrane protein named caveolin. We compared the freeze-fracture behavior of caveolae in glutaraldehyde-fixed and cryofixed mouse fibroblast cells and found distinct differences. In glutaraldehyde-fixed cells almost all caveolae were cross-fractured through their pore and only very few caveolar membranes were membrane-fractured. We found the reverse situation in rapid frozen cells without any chemical fixation where most of the caveolae were membrane-fractured, showing different degrees of invagination from nearly flat to deeply invaginated. In ultrathin sections of glutaraldehyde-fixed heart endothelial cells, caveolae exhibit the well known omega-like shape. In high-pressure frozen, freeze-substituted and low temperature embedded heart endothelial cells, the caveolae frequently exhibit a cup-like shape without any constriction or pore. The cup-like caveolar shape could also be shown by tilt series analysis of freeze-fracture replicas obtained from cryofixed cells. Freeze-fracture immunolabeling of caveolin-1 revealed a lateral belt-like caveolin alignment. These findings point out that the constricted "neck" region of caveolae in most cases is an effect that is caused and intensified by the glutaraldehyde fixation. Our data indicate that caveolae in vivo show all degrees of invagination from nearly flat via cup-like depressed to in a few cases omega-like.

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Improved antigen retrieval in freeze-fracture cytochemistry by evaporation of carbon as first replication layer

Cited by 14 publications

References 46 publications

Quantitative retention of membrane lipids in the freeze-fracture replica

Quantitative retention of membrane lipids in the freeze-fracture replica

Heterotypic gap junctions at glutamatergic mixed synapses are abundant in goldfish brain

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation

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