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Immunohistochemistry localises myosin-7a to cochlear efferent boutons

Citation

Sirko, Piotr; Kozlov, Andrei (2021), Immunohistochemistry localises myosin-7a to cochlear efferent boutons, Dryad, Dataset, https://doi.org/10.5061/dryad.9s4mw6mhz

Abstract

Myosin 7a is often used to visualise sensory hair cells due to its well-characterised and localised expression profile. We thus conducted myosin-7a immunostaining across all 3 turns of the adult rat organ of Corti to visualise hair cells. As expected, we observed myosin 7a staining in both inner and outer hair cells. Unexpectedly, we also observed strong myosin 7a staining in the medial olivocochlear efferent synaptic boutons contacting the outer hair cells. Efferent bouton myosin-7a staining was present across all 3 turns of the cochlea. We verified this localisation by co-staining with a known efferent bouton marker, the vesicular acetylcholine transporter. 

Methods

A Leica SP5 upright confocal microscope with two-photon imaging capabilities was used to image the exposed and stained organs of Corti. Cochleas were superglued to the lids of 55-mm diameter cell culture Petri dishes prior to imaging to ensure the organ of Corti would be in an appropriate orientation and immersed in PBS. The Argon 488, Diode 543, Diode 594 and Diode 633 were used for single-photon excitation of the Alexa dyes conjugated to the secondary antibodies and the Mai Tai eHP DeepSee 5332 laser set to a wavelength of 800 nm was used for two-photon excitation of the phalloidin conjugated Alexa 405. A 25x/0.95NA water immersion objective (HCX IRAPO L25x/0.95 W) was used. Images were recorded in 12 bits at a resolution of 1024 x 1024 and further appropriate Look Up Table colors and processed in ImageJ (https://imagej.nih.gov/ij/). Methods are described in detail in the publication "Immunohistochemistry localises myosin-7a to cochlear efferent boutons" which has been conditionally accepted for publication in the journal Welcome Open Research. Article DOI will be linked to dataset once published. 

Usage Notes

Data is in the tiff file format, which can be opened using ImageJ (https://imagej.nih.gov/ij/) or any other program with similar capabilities. Data has been zipped up for easier download and organised in the zip file according to which figure it is associated within the manuscript. Labelling of individual files reflects the stainings, turn of the rat cochlea from which it has been acquired (base, mid, apex), rat from which it was acquired and/or cochlea of the rat, if relevant.