RESUMO
Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to <20 µm as formalin fixed paraffin section on glass slides or <500 µm for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples >500 µm using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth >150 µm with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.
