Comparing and Optimizing Tissue Clearing Protocols for Cochlear Immunostaining and 3D Reconstruction by Light-Sheet...

Current standard methods for immunohistochemical analysis of the inner ear rely on cryosectioning of the intact cochlea or dissection of the organ of Corti. Cryosections provide only limited structural information and do not allow quantification of hair cell numbers per basilar membrane length, whereas dissections require substantial technical expertise and often result in damage to the basal cochlear turns. Tissue clearing has largely overcome these limitations and has been widely applied to organs such as the brain, heart, liver, and kidney. However, despite several reports describing cochlear clearing, a systematic comparison of diverse methods is lacking.

Here, we present a comparative evaluation of five established clearing protocols applied to the mouse cochlea, including three organic solvent-based (applied to n = 10 cochleae each) and two aqueous-based approaches (n = 4/8).

Among these, iDISCO/cDISCO (i/cDISCO) and ethyl cinnamate (ECi) provided superior tissue transparency, morphological preservation, and immunofluorescence staining quality. In contrast, PEGASOS resulted in reduced transparency and occasional wave-like distortion of hair cell rows, probably due to uneven tissue shrinkage. ScaleS and FRUIT exhibited limited clearing performance and variable staining results. Based on these findings, we optimized the two best-performing protocols to enable cochlear clearing within only five days, using inexpensive and minimally toxic reagents.

The resulting fastDISCO and fastECi protocols achieved clearing and immunolabeling quality comparable to their full-length counterparts while preserving tissue morphology (applied to n = 17/20 cochleae). These rapid clearing protocols support reproducible 3D light-sheet microscopy of immunostained cochleae and enable automated cell segmentation and quantitative analysis of distinct cell types, including the inner and outer hair cells.