Premium
Confocal microscopy of thick sections from acrylamide gel embedded embryos
Author(s) -
Germroth Patricia G.,
Gourdie Robert G.,
Thompson Robert P.
Publication year - 1995
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.1070300608
Subject(s) - confocal microscopy , microscopy , confocal , acrylamide , embryo , materials science , chemistry , biophysics , biology , microbiology and biotechnology , optics , composite material , physics , polymer , monomer
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 μm. 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. © 1995 Wiley‐Liss, Inc.