Multicolor 3D Confocal Imaging of Thick Tissue Sections

In multicellular organisms, most physiological and pathological processes involve an interplay between various cells and molecules that act both locally and systemically. To understand how these complex and dynamic processes occur in time and space, imaging techniques are key. Advances in tissue processing techniques and microscopy now allow us to probe these processes at a large scale and at the same time at a level of detail previously unachievable. Indeed, it is now possible to reliably quantify multiple protein expression levels at single-cell resolution in whole organs using three-dimensional fluorescence imaging techniques. Here we describe a method to prepare adult mouse bone tissue for multiplexed confocal imaging of thick tissue sections. Up to eight different fluorophores can be multiplexed using this technique and spectrally resolved using standard confocal microscopy. The optical clearing method described allows detection of these fluorophores up to a depth of >700 μm in the far-red. Although the method was initially developed for bone tissue imaging, we have successfully applied it to several other tissue types.