Decalcification by ascorbic acid for immuno- and affinohistochemical techniques on the inner ear

Abstract

An ascorbic acid decalcifying solution was applied to immuno- and affinohistochemical studies on the inner ear. Rat inner ears fixed in 4% paraformaldehyde in PBS or in 2% acetic acid in ethanol solutions were adequately decalcified in an ascorbic acid solution, at a temperature of 4 degrees C. The decalcifying solution was prepared with 1% ascorbic acid and 0.84% sodium chloride in distilled water (pH 2.5-2.6). The decalcification time was in a direct relationship to the specimen calcification. In this study, two neuroactive substances (gamma-aminobutyric acid and calcitonin gene-related peptide), neurofilaments, and the galectine endogenous lectin were successfully detected immunohistochemically.

Gap junctions in the inner ear: Comparison of distribution patterns in different vertebrates and assessement of connexin composition in mammals

“…For whole‐mount preparations, and for frozen sections from guinea pigs and gerbils, the tissues were then dissected out under phosphate‐buffered saline (PBS). For frozen sections from the mice, the bullae were first decalcified in either 4.13% EDTA, pH 7.5, for 2 days at 4°C or 1% ascorbic acid, 0.8% NaCl for 7 days, with changes of solution every 2 days (Merchan‐Perez et al, 1999). Samples for frozen sections were incubated overnight in 30% sucrose in PBS, then embedded in 1% low‐gelling‐temperature agarose in 18% sucrose in PBS at 37°C.…”

Electrophysiological monitoring of hearing function during cochlear perilymphatic perfusions

“…Then, each cochlea was gently removed and quickly post fixed in the same solution for 12 h at 4 C. Afterwards, each cochlea was washed and decalcified in an ascorbic acid solution (1% in NaCl 0.9%) [17] for 10 days. Post fixation was carried out with osmic acid (4% in a phosphate solution).…”