title: Immunohistochemical detection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP).
abstract: Traditional histochemical detection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) can impose substantial technical limitations on studies requiring co-localization of neurotransmitters, receptors and other neural antigens. The goal of our experiments was to establish the ideal conditions and reagents for immunohistochemical detection of WGA-HRP. WGA-HRP was injected into the tongues and vibrissae pads of adult rats to characterize labeling of somas and synapses, respectively. Rats were perfused with either 4% paraformaldehyde (for light microscopy, LM) or 4% paraformaldehyde/0.15% glutaraldehyde (for electron microscopy, EM) after survival times of 2, 3, 4, 5 or 6 days. For LM, brainstem tissue was cut on a cryostat at 20 microm and collected onto glass slides. For EM, tissue was sectioned with a vibratome at 50 microm and processed free floating. For LM, WGA-HRP was detected with goat anti-HRP, goat anti-WGA, biotinylated goat anti-HRP or biotinylated goat anti-WGA antibodies. For EM, WGA-HRP was detected with biotinylated goat anti-WGA and anti-HRP antibodies. Survival intervals of 3 days were ideal for staining of hypoglossal neurons, whereas an interval of 4 days produced the strongest staining of synapses within the spinal trigeminal nucleus. For LM, the biotinylated antibodies resulted in better signal-to-noise ratios than the unconjugated antibodies. At both the LM and EM levels, the biotinylated antibody to WGA produced better quality staining than the biotinylated antibody to HRP.
authors.affiliation: Departments of Neurology and Anatomy and Neurobiology, University of Tennessee Health Science Center, 855 Monroe Avenue, Suite 415, Memphis, TN 38163, USA.
authors.forename: Suzhen
authors.initials: S
authors.lastname: Gong
authors.forename: Mark S
authors.initials: MS
authors.lastname: LeDoux
mesh_terms: D000818:Animals; D000906:Antibodies; D019921:Biotinylation / Q000379:methods; D001933:Brain Stem / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D004817:Epidermis; D007150:Immunohistochemistry / Q000379:methods; D007267:Injections; D008657:Metabolic Clearance Rate; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure*; D011786:Quality Control; D051381:Rats; D013194:Staining and Labeling / Q000379:methods; D013569:Synapses / Q000378:metabolism / Q000648:ultrastructure*; D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate / Q000378:metabolism*
chemical_list: D000906:Antibodies; D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate