Hydroxypropyl Methacrylate, a New Water-Miscible Embedding Medium for Electron Microscopy

• Published in: The Journal of cell biology Vol. 26; no. 1; pp. 137 - 155
• Main Authors: Leduc, Elizabeth H., Holt, S. J.
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01.07.1965; The Rockefeller University Press
• Subjects: Acetates; Acrylic Resins; Animals; Cells; Chromatin; Dehydration; Deoxyribonucleases; Diaphragm - anatomy & histology; Duodenum - anatomy & histology; Electron microscopy; Enzymes; Histocytochemistry; Intestines; Kidney - anatomy & histology; Liver; Liver - anatomy & histology; Methacrylates; Microscopy, Electron; Pancreas - anatomy & histology; Polymerization; Rats; Ribonucleases
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.26.1.137

Aldehyde-fixed rat tissues were variously dehydrated and impregnated in water-miscible 2-hydroxypropyl methacrylate (HPMA) containing 3 to 20 per cent water and 0.1 per cent α,α-azobisisobutyronitrile as catalyst for subsequent polymerization with ultraviolet light. Heat polymerization was also effective. Blocks of embedded tissue readily gave ultrathin sections, which required staining by uranyl acetate and/or lead stains to give adequate contrast for electron microscopy. The ultrastructure of pancreas, kidney, muscle, and intestine was well preserved by aldehyde fixation alone. Use of postfixation in osmium tetroxide or direct osmium tetroxide fixation was unsatisfactory. The fine structure of aldehyde-fixed liver from fasted rats was well preserved, whereas that from normal rats showed considerable disorganization and collapse, apparently because of extraction of glycogen during the embedding procedure. Enzymatic extraction of proteins by pepsin and of ribonucleic acid by ribonuclease after either formaldehyde or glutaraldehyde fixation was rapidly effected by direct treatment of ultrathin sections with solutions of the enzymes. In contrast, no digestion of chromatin by deoxyribonuclease could be detected. In spite of this present limitation, HPMA appears to have several advantages over earlier water-miscible embedding media for electron microscopy and to be particularly suitable for ultrastructural cytochemistry.