Light and electron microscopical demonstration of concanavalin A and wheat-germ agglutinin binding sites by use of antibodies against the lectin or its label (peroxidase).
Three straining protocols for the ultrastructural visualization of concanavalin A (ConA) and wheat germ agglutinin (WGA) binding sites were applied to samples of nervous tissue embedded in Lowicryl K4M. The hypothalamo-neurohypophysial neurosecretory system was chosen for this investigation because it has two major neuronal populations, one secreting vasopressin, whose precursor is glycosylated, and the other secreting oxytocin whose precursor form is not glycosylated. The series of incubations of the tissue sections for the three protocols were: Protocol 1: i) non labeled ConA or WGA; ii) ConA or WGA antibody; iii) protein A-gold; Protocol 2: i) pre-prepared WGA-anti-WGA complex; ii) protein A-gold; Protocol 3: i) peroxidase-labeled ConA or WGA; ii) anti-peroxidase; iii) protein A-gold. The three methods allowed to detect fine differences in the distribution of sugar residues. This, in turn, made it possible to distinguish vasopressin granules containing precursor forms from those containing processed precursor. At the light microscopic level the three methods were successfully applied to paraffin and 1-micron methacrylate sections by using a second antibody, PAP complex and the diaminobenzidine reaction.
Peruzzo
B
B
Instituto de Histología y Patología, Facultad de Medicina, Universidad Austral de Chile, Valdivia.
Rodríguez
E M
EM
D000818:Animals; D003208:Concanavalin A / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007030:Hypothalamo-Hypophyseal System / Q000166:cytology / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry / Q000379:methods*; D010544:Peroxidases / Q000378:metabolism*; D051381:Rats; D011975:Receptors, Mitogen / Q000378:metabolism*; D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; D014909:Wheat Germ Agglutinins / Q000378:metabolism*
D011975:Receptors, Mitogen; D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; D014909:Wheat Germ Agglutinins; D003208:Concanavalin A; D006735:Horseradish Peroxidase; D010544:Peroxidases