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In situ freeze‐fracture of monolayer cell cultures grown on a permeable support
Author(s) -
Todd John H.,
Sens Donald A.,
Sens Mary Ann,
HazenMartin Debra
Publication year - 1992
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.1070220308
Subject(s) - paracellular transport , monolayer , polyvinyl alcohol , tight junction , transcellular , membrane , ultrastructure , intramembranous ossification , materials science , semipermeable membrane , fracture (geology) , biophysics , chemistry , composite material , microbiology and biotechnology , anatomy , nanotechnology , biology , permeability (electromagnetism) , biochemistry
The growth of cultured epithelial cells on permeable supports allows increased cell differentiation and the assessment of a variety of transcellular and paracellular transport processes. The need to assess the corresponding ultrastructural characteristics of these cells underidentical conditions prompted this laboratory to develop a reliable method for producing freezefracture replicas of these cultures. Sections of filter inserts with the cell‐side facing up are placed between layers of polyvinyl alcohol with a strip of mylar positioned on the upper layer of polyvinyl alcohol. Following freezing, the monolayer is fractured by lifting the mylar strip from the assembly. The result is a consistent fracture of the apical membrane sufficient for analysis of tight junction sealing strands, microvilli distribution, and intramembranous particle (IMP) distribution between apical and lateral membrane domains. This method utilizes standard equipment and readily available materials and, most importantly, allows the freeze‐fracture and replication of an undisturbed cell monolayer. © 1992 Wiley‐Liss, Inc.