FAST, NON-TOXIC, CLEARED TISSUE SAMPLE PREPARATION FOR HIGH-THROUGHPUT LIGHT SHEET IMAGING AND ANALYSIS

Whole-organ light-sheet fluorescence microscopy (LSFM) enables rapid, high-resolution imaging of intact tissue while preserving its three-dimensional architecture. In auditory research, decalcification, labelling, and optical clearing of the cochleae allow comprehensive visualization of cochlear structures across large volumes. As a result, LSFM provides powerful insights into cochlear development, morphology, pathology, and therapeutic outcomes. There have been many advancements in LSFM imaging and volumetric image analysis, while the whole-organ sample preparation has remained mainly unchanged, with long processing times and the use of harmful solutions. To address this challenge, we introduce a fast, non-toxic whole-organ cochlear LS sample preparation and staining method, which we term Microwave fast immunohistochemistry (MW-fIHC). MW-fIHC utilizes regular non-toxic immunohistochemistry reagents and a PELCO BioWave® Pro+ sample processing system to reliably decalcify, label, and clear the mouse cochlea within 2 weeks. Combined with fast high-resolution isotropic light sheet imaging (Aakhte et al, 2025) and automated segmentation of spiral ganglion neurons (SGNs), inner hair cells (IHC), and their ribbon synapses using CochleaNet (Roos&Diniz et al, 2025, BioRxiv), this protocol presents a workflow from sample to data at an unprecedented speed and throughput. To demonstrate the applicability of 2-week MW-fIHC, we compared it to conventional 6-week iDISCO+ for mouse cochleae immunolabelled for SGNs, IHC, and their synapses. Moreover, we demonstrated and compared the use of PELCO BioWave® Pro+ with iDISCO reagents to increase staining efficiency and sample throughput in a 3-week protocol.