Characterization of the Mucomicrovillar Complex in the Vomeronasal Epithelium with Lectinohistochemistry and Confocal Laser Scanning Microscopy

Abstract

Lectinohistochemistry and confocal laser scanning microscopy (CLSM) were utilized to resolve the mucomicrovillar complex (MMC) [1] of the vomeronasal sensory epithelium (VE) into mucoid and sensory components, and to identify glycoconjugates in these components. Six-week-old Sprague-Dawley rats of both sexes were anesthetized deeply with Nembutal and transcardially perfused with Zamboni’s fixative. The vomeronasal organ (VNO), olfactory mucosa (OM), and the septal organ of Masera (SO) were sectioned serially in the transverse plane, using a cryostat. Binding sites for the isolectin Bandeiraea simplicifolia-I B4 (BS-I-B4), which specifically recognizes terminal α-d-galactose (α-Gal) sugar residues [2], were localized on tissue sections with the standard fluorescence technique for fluorescein isothiocyanate (FITC)-BS-I-B4, and the avidinbiotin-peroxidase complex (ABCperoxidase) technique for biotin-conjugated BS-I-B4. Some sections were double-labeled with the antibody against olfactory marker protein (OMP) using the ABC-fluorochrome (Texas Red) technique and with FITC-BS-I-B4 using the fluorescence technique. These sections were analyzed with a confocal laser scanning microscope (MultiProbe 2001; Molecular Dynamics, Sunnyvale, Calif.), using a filter set for simultaneous detection, that is, dual scanning, of the optimal emission spectrum of FITC (peak, 520 nm) and Texas Red (peak, 620 nm).