A method for using low-temperature embedding media for electron microscopy of cells grown on microporous supports.
Attempts were made to embed Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose microporous supports in Lowicryl, a medium designed for the retention of antigenicity for electron microscopic immunocytochemical studies. It was found that the membrane fragmented during the prescribed embedding procedure leading to loss of the cell sample. This necessitated the formulation of a new combination of fixative and dehydration agents which would allow: (i) preservation of membrane and cellular integrity; (ii) infiltration and embedding in Lowicryl; and (iii) detection of a specific antigen in thin sections. The cellular monolayer and organelle profiles were best preserved with glutaraldehyde fixation at room temperature followed by ethanol dehydration. Since the latter was carried out at temperatures attainable with an ice-salt bath, there was no need for a special ultralow-temperature apparatus. This procedure was applied to a MDCK cell clone that consisted of stable secretors of human growth hormone (hGH), as a result of being transfected with a plasmid containing the hGH gene. Thus, it was demonstrated that hGH could be detected by immunogold labeling of thin sections and localized to specific cellular structures. The procedure developed in this report is applicable to cells grown on two other supports and may be extended further.
Rudick
V L
VL
Department of Anatomy and Cell Biology, Texas College of Osteopathic Medicine, Fort Worth 76107.
Brun-Zinkernagel
A M
AM
Rudick
M J
MJ
Chakraborty
S
S
D000818:Animals; D002460:Cell Line; D004285:Dogs; D005455:Fluorescent Antibody Technique; D013006:Growth Hormone / Q000032:analysis; D006801:Humans; D007382:Intermediate Filaments / Q000276:immunology; D008566:Membranes; D016253:Microscopy, Immunoelectron / Q000379:methods*; D013696:Temperature; D016610:Tissue Embedding / Q000379:methods*
D013006:Growth Hormone