Use of LR white resin for post-embedding immunolabelling of brain tissue.
The object of this study was to investigate the applicability of the acrylic resin 'LR White' to immunolabelling of various antigenic determinants in aldehydefixed rat CNS tissue. Antibodies were used, which worked well in paraffin sections and therefore were suitable to detect antigens resistant to complete dehydration and heat. Different LR White embedding protocols were employed in order to select the preparation conditions that adequately preserved both the antigenicity and fine structure. Specimens were completely dehydrated with up to 100% ethanol, which was followed by various infiltration times with LR White monomer. Polymerization of the resin was induced by heat, a chemical catalytic procedure (accelerator), or ultraviolet (UV) light. Paraffin, as well as semithin and ultrathin LR White sections were incubated with antibodies reacting to antigens located on the cell surface (stage-specific embryonic antigen-1; SSEA-1), within the plasma membrane (myelin basic protein), in the cytosol (HNK-1, S100 protein), in the cytoskeleton (GFAP, vimentin, neurofilament protein, INT-FIL), and in the extracellular matrix (laminin). All of the examined antigens were immunocytochemically detectable in paraffin-embedded material, while the carbohydrate moieties, HNK-1 and SSEA-1, were not immunoreactive in LR White sections. However, in cryostat sections processed for pre-embedding immunoelectron microscopy, the HNK-1 epitope and SSEA-1 were immunolabelled. Polymerization carried out under UV light led to better structural preservation of brain tissue than resin cured with heat or catalyst. The length of prior infiltration with monomer apparently had no effect on tissue preservation. Consequently, UV light-induced polymerization of LR White gives acceptable morphology of brain tissue. However, the use of this acrylic resin is restricted to the detection of some CNS antigens only.
Gocht
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Anatomisches Institut, Abteilung für Neuroanatomie, Universität Hamburg, BRD.
D000180:Acrylic Resins; D000818:Animals; D000941:Antigens / Q000032:analysis / Q000187:drug effects; D001921:Brain / Q000033:anatomy & histology* / Q000276:immunology; D018959:CD57 Antigens / Q000032:analysis; D016616:Cryoultramicrotomy; D003681:Dehydration; D006358:Hot Temperature; D007150:Immunohistochemistry / Q000379:methods; D016256:Lewis X Antigen / Q000032:analysis; D008297:Male; D008854:Microscopy, Electron; D051381:Rats; D017208:Rats, Wistar; D016610:Tissue Embedding / Q000379:methods*; D014466:Ultraviolet Rays
D000180:Acrylic Resins; D000941:Antigens; D018959:CD57 Antigens; D016256:Lewis X Antigen; C048707:LR white