Improved antigen retrieval in freeze-fracture cytochemistry by evaporation of carbon as first replication layer.
The recently developed freeze-fracture replica immunolabeling technique uses sodium dodecyl sulfate to clean replicas obtained from chemically unfixed, rapidly frozen cells by evaporation of platinum as first and carbon as second replication layer. The detergent dissolves remains of cellular material with the exception of components which are in direct contact to the replica film. Membrane lipids and membrane protein complexes of the protoplasmic and the exoplasmic membrane halves remain attached to the replica film and are accessible for cytochemical localization. We immunolabeled the membrane proteins caveolin-1 and connexin 43 in mouse cell lines as well as the membrane attached protein tetrachloroethene reductive dehalogenase (PceA) in bacterial cells at freeze-fracture replicas generated by different evaporation parameters. The labeling experiments for caveolin-1 and the PceA showed that freeze-fracture replication of cellular membranes accomplished with thin platinum layers as well as replication with carbon as first evaporation layer lead in these cases to an improved antigen retrieval, whereas the labeling efficiency of connexin 43 was not affected by different evaporation conditions.
Schlörmann
Wiebke
W
Elektronenmikroskopisches Zentrum, Klinikum der Friedrich-Schiller-Universität, Ziegelmühlenweg 1, D-07743, Jena, Germany.
John
Markus
M
Steiniger
Frank
F
Westermann
Martin
M
Richter
Walter
W
D000818:Animals; D000941:Antigens / Q000032:analysis* / Q000276:immunology; D002244:Carbon / Q000737:chemistry*; D051242:Caveolin 1 / Q000032:analysis / Q000276:immunology; D018031:Connexin 43 / Q000032:analysis / Q000276:immunology; D005614:Freeze Fracturing / Q000379:methods*; D017612:Gold Compounds; D006651:Histocytochemistry / Q000379:methods*; D051379:Mice; D041681:NIH 3T3 Cells / Q000276:immunology / Q000378:metabolism
D000941:Antigens; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D018031:Connexin 43; D017612:Gold Compounds; D002244:Carbon