A new immunohistochemical method for the detection of gentamicin in inner ear fluid compartments

Abstract

A new method was developed for frozen section detection of antigens that natively occur in the cochlear peri- and endolymph. A combination of immuno-histochemistry and immunoblot assay enabled topological and quantitative detection of small and hydrophilic molecules (such as the aminoglycoside antibiotics) in frozen sections of the inner ear compartments (scala tympani, scala vestibuli and cochlear duct). A selective localization is possible in the peri- and endolymphatic region of each coil of the cochlea. During sectioning of the cochlea, a small piece of a nitrocellulose membrane is placed to the surface of the intersection and briefly warmed. The sections are cut, simultaneously attached to a nitrocellulose membrane on which the aminoglycoside antibiotics remain adsorbed without any fixation procedure. Using this method, immunoincubation to detect gentamicin was performed in a way usually done in western blot analysis. Results with two different enzyme reactions with the enzyme conjugated to a second antibody (i.e., dye as substrate and the chemiluminescence detection system) are presented and compared. This histoimmunoblot assay provides a general non-radioactive and sensitive immunohistochemical tool for the localization of compounds occurring in extracellular body fluid compartments. For inner ear research this method now enables the investigation of the penetration and distribution of therapeutics in peri- and endolymphatic sites and can even be applied to separately quantifying concentrations of a substance in different coils of the same cochlear section.

Multiple Antibiotic Resistance in Arabidopsis Is Conferred by Mutations in a Chloroplast-Localized Transport Protein

“…To date, there is no report on uptake studies of aminoglycoside antibiotics in a plant system; therefore, no convenient assay was available. The assay developed here allows for inexpensive, nonradioactive detection of antibiotic and is based on the ability of aminoglycosides to adsorb onto nitrocellulose membrane without the need for fixation (Mihelic-Rapp and Giebel, 1996). Differences are clearly seen in lysate spots from mar1-1 mutant chloroplasts, as compared to wild-type chloroplasts and chloroplasts from an overexpression line (Fig.…”

Comparative analysis of combination kanamycin-furosemide versus kanamycin alone in the mouse cochlea

“…The mechanism of uptake of aminoglycosides from the vascular space into sensory cells has also been carefully studied, and investigators have demonstrated the presence of aminoglycosides in a number of cochlear cell types including the sensory cells of the cochlea and the stria vascularis, spiral ligament, and spiral limbus (Dai and Steyger, 2008; de Groot et al, 1990; Hashino et al, 2000; Mihelic-Rapp and Giebel, 1996). Certain cells that demonstrate uptake of the drug are susceptible whereas others can absorb the drug and remain resistant.…”