CONFOCAL MICROSCOPY OF THICK SECTIONS FROM ACRYLAMIDE-GEL EMBEDDED EMBRYOS

The preparation of optically clear, thick sections of fragile embryoni c tissues greatly aids the power of confocal scanning laser microscopy in imaging three-dimensional structures. We report here conditions fo r embedding, sectioning, and staining embryos in polyacrylamide gels f or a variety of confocal imaging techniques. Infiltration of tissues i n standard mixtures of 10-15% acrylamide monomer yields, upon polymeri zation, blocks that cut easily by vibratome between 50 and 1,000 mu m. These conditions worked well for tissues previously stained or for st aining gel sections with low molecular weight water-soluble fluorochro mes (MW < 5 kD [e.g., propidium iodide, phalloidin]). For immunostaini ng of tissue after embedding and sectioning, the acrylamide concentrat ion was reduced to 2-3% acrylamide to allow access of immunoglobulins to antigenic sites; such gels were supplemented with 1% agarose to fac ilitate sectioning and handling. Either method yielded abundant, optic ally clear, and easily handled sections for mounting and examination i n water-miscible media. (C) 1995 Wiley-Liss, Inc.