Confocal microscopy of thick sections from acrylamide gel embedded embryos.
The preparation of optically clear, thick sections of fragile embryonic tissues greatly aids the power of confocal scanning laser microscopy in imaging three-dimensional structures. We report here conditions for embedding, sectioning, and staining embryos in polyacrylamide gels for a variety of confocal imaging techniques. Infiltration of tissues in standard mixtures of 10-15% acrylamide monomer yields, upon polymerization, blocks that cut easily by vibratome between 50 and 1,000 microns. These conditions worked well for tissues previously stained or for staining gel sections with low molecular weight water-soluble fluorochromes (MW < 5 kD [e.g., propidium iodide, phalloidin]). For immunostaining of tissue after embedding and sectioning, the acrylamide concentration was reduced to 2-3% acrylamide to allow access of immunoglobulins to antigenic sites; such gels were supplemented with 1% agarose to facilitate sectioning and handling. Either method yielded abundant, optically clear, and easily handled sections for mounting and examination in water-miscible media.
Germroth
P G
PG
Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston 29425, USA.
Gourdie
R G
RG
Thompson
R P
RP
D000180:Acrylic Resins; D000818:Animals; D015820:Cadherins / Q000032:analysis; D015816:Cell Adhesion Molecules, Neuronal / Q000032:analysis; D002642:Chick Embryo; D004622:Embryo, Mammalian / Q000737:chemistry / Q000166:cytology*; D016326:Extracellular Matrix Proteins / Q000032:analysis; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006321:Heart / Q000196:embryology; D018613:Microscopy, Confocal; D008867:Microtomy; D009206:Myocardium / Q000737:chemistry / Q000166:cytology; D012377:Rodentia; D013552:Swine; D019063:Tenascin; D016610:Tissue Embedding
D000180:Acrylic Resins; D015820:Cadherins; D015816:Cell Adhesion Molecules, Neuronal; D016326:Extracellular Matrix Proteins; D005456:Fluorescent Dyes; D019063:Tenascin; C016680:polyacrylamide gels