Postembedding light- and electron microscopic immunocytochemistry of amino acids: description of a new model system allowing identical conditions for specificity testing and tissue processing

Abstract

Specificity testing should be performed under conditions identical to or closely similar to those of the immunocytochemical procedure. This paper describes a new model system that meets this requirement for postembedding immunocytochemistry of amino acids at the light- and electron microscopic levels. Test conjugates, obtained by reacting different amino acids with brain macromolecules in the presence of glutaraldehyde, were freeze-dried and embedded in an epoxy resin (Durcupan) exactly as for brain tissue. One section from each of the embedded amino acid conjugates and from a brain protein-glutaraldehyde conjugate (without amino acid) were piled on top of each other and embedded anew. Transverse semithin (0.5 micron) and ultrathin sections were cut through the stack. These test sections, in which all the different conjugates were represented, were then processed in the same drops of sera as the tissue sections to permit identical conditions for testing and immunocytochemistry. After immunogold labelling for electron microscopy, a quantitative expression of crossreactivity was obtained by computer-assisted calculation of gold particle densities over the different conjugates. The antisera used in the present study (glutamate antiserum 13, taurine antiserum 20, and GABA antiserum 26) showed highly selective labelling of the respective amino acid conjugates and produced distinct labelling patterns in simultaneously processed cerebellar sections.

Cervicothalamic tract terminals are enriched in glutamate-like immunoreactivity: an electron microscopic double-labeling study in the cat

“…The antisera used have previously been screened in "spot tests" against brain macromolecule-glutaraldehyde complexes of more than 40 different small molecules endogenous to the brain and found to react selectively with fixed Glu and Gln, respectively (Ottersen and Storm-Mathisen, 1984;Laake et al, 1986). The specificity of the postembedding immunogold labeling was monitored in the present experiments by parallel incubation of "control sections" containing Durcupan-embedded brain macromolecule-glutaraldehyde fixation complexes of the six most abundant amino acids in the brain (Ottersen, 1987).…”

Redistribution of Glutamate and Glutamine in Slices of Human Neocortex Exposed to Combined Hypoxia and Glucose Deprivation in vitro

“…Ultrathin sections (obtained from the superficial part of the slices) were treated according to a postembedding immunogold procedure with a polyclonal antiserum (specifically recogniz ing fixed glutamate or glutamine) followed by a secondary antibody coupled to colloidal gold particles. Detailed de scriptions of the production and characterization of the antisera have been published previously (Storm-Mathisen et aI., 1983;Otters en and Storm-Mathisen, 1984;Laake et al, 1986;Ottersen, 1987Ottersen, , 1989aJi et aI., 1991).…”

“…The first protocol was modified from Somogyi and Hodgson (1985) and has been described in detail previ ously (Ottersen, 1987(Ottersen, , 1989a. Following treatment with 1% HI04 (7 min) and 9% NaHI04 (15 min) to alleviate the masking effect of OS04' the sections were incubated for 2 h in the primary antiserum to glutamate (code no.…”

“…monitored by including test sections containing conju gates of the most abundant amino acids in the brain, as described by Ottersen (1987). These test sections accom panied the tissue sections throughout the immunocyto chemical procedure.…”

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

“…Ottersen, 1987;Holmseth et al, 2006;Lorincz and Nusser, 2008;Rhodes and Trimmer, 2006). Virtually all assay conditions can affect antibody binding, including protein conformation and hydrophobic interactions (e.g.…”