An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry. 1995

K D Phend, and A Rustioni, and R J Weinberg
Department of Cell Biology & Anatomy, University of North Carolina, Chapel Hill 27599.

Immunocytochemistry for amino acids with post-embedding gold is compatible with glutaraldehyde fixation, osmication, and embedding in epoxy-based plastics, but immunogold detection of larger molecules in the central nervous system commonly requires special procedures, e.g. minimizing exposure to glutaraldehyde, eliminating osmium, cryosectioning, and/or embedding in acrylic plastics. These make samples more difficult to prepare and view and may compromise structural preservation. We report a new technique, fixing with high levels of glutaraldehyde, replacing osmium with tannic acid followed by other heavy metals and p-phenylenediamine, and embedding in Epon. This method optimizes antigenicity while retaining the structural preservation and convenient handling of standard embedding techniques. Compared to standard Epon embedment, labeling for neuropeptides in brain and spinal cord is improved. Moreover, the present method yields excellent labeling of glutamate receptors (difficult to identify with traditional post-embedding techniques) and enables simultaneous visualization of associated neurotransmitters.

UI MeSH Term Description Entries
D008297 Male Males
D009478 Neuropeptide Y A 36-amino acid peptide present in many organs and in many sympathetic noradrenergic neurons. It has vasoconstrictor and natriuretic activity and regulates local blood flow, glandular secretion, and smooth muscle activity. The peptide also stimulates feeding and drinking behavior and influences secretion of pituitary hormones. Neuropeptide Y-Like Immunoreactive Peptide,Neuropeptide Tyrosine,Neuropeptide Y Like Immunoreactive Peptide,Tyrosine, Neuropeptide
D009479 Neuropeptides Peptides released by NEURONS as intercellular messengers. Many neuropeptides are also hormones released by non-neuronal cells. Neuropeptide
D009992 Osmium A very hard, gray, toxic, and nearly infusible metal element, atomic number 76, atomic weight 190.2, symbol Os.
D011309 Preservation, Biological The process of protecting various samples of biological material. Biological Preservation,Preservation, Biologic,Biologic Preservation
D001921 Brain The part of CENTRAL NERVOUS SYSTEM that is contained within the skull (CRANIUM). Arising from the NEURAL TUBE, the embryonic brain is comprised of three major parts including PROSENCEPHALON (the forebrain); MESENCEPHALON (the midbrain); and RHOMBENCEPHALON (the hindbrain). The developed brain consists of CEREBRUM; CEREBELLUM; and other structures in the BRAIN STEM. Encephalon
D004853 Epoxy Resins Polymeric resins derived from OXIRANES and characterized by strength and thermosetting properties. Epoxy resins are often used as dental materials. Epoxy Resin,Resin, Epoxy,Resins, Epoxy
D005680 gamma-Aminobutyric Acid The most common inhibitory neurotransmitter in the central nervous system. 4-Aminobutyric Acid,GABA,4-Aminobutanoic Acid,Aminalon,Aminalone,Gammalon,Lithium GABA,gamma-Aminobutyric Acid, Calcium Salt (2:1),gamma-Aminobutyric Acid, Hydrochloride,gamma-Aminobutyric Acid, Monolithium Salt,gamma-Aminobutyric Acid, Monosodium Salt,gamma-Aminobutyric Acid, Zinc Salt (2:1),4 Aminobutanoic Acid,4 Aminobutyric Acid,Acid, Hydrochloride gamma-Aminobutyric,GABA, Lithium,Hydrochloride gamma-Aminobutyric Acid,gamma Aminobutyric Acid,gamma Aminobutyric Acid, Hydrochloride,gamma Aminobutyric Acid, Monolithium Salt,gamma Aminobutyric Acid, Monosodium Salt
D005976 Glutaral One of the protein CROSS-LINKING REAGENTS that is used as a disinfectant for sterilization of heat-sensitive equipment and as a laboratory reagent, especially as a fixative. Glutaraldehyde,Cidex,Diswart,Gludesin,Glutardialdehyde,Glutarol,Korsolex,Novaruca,Sekumatic,Sonacide,Sporicidin
D006652 Histological Techniques Methods of preparing tissue for examination and study of the origin, structure, function, or pathology. Histologic Technic,Histologic Technics,Histologic Technique,Histologic Techniques,Histological Technics,Technic, Histologic,Technics, Histologic,Technique, Histologic,Techniques, Histologic,Histological Technic,Histological Technique,Technic, Histological,Technics, Histological,Technique, Histological,Techniques, Histological

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