Use of the CLARITY technique for identifying fluorescent markers on thick slices of spinal cord

Abstract

The study of the interconnections established between neurons themselves and with glial cells is one of the major challenges of neuroscience. Fluorescent antibodies allow identification and localization of different tissue structures. However, their efficient employment requires the use of thin sections of samples, which constitute a limitation for the study of nervous system interconnections. Likewise, increased thickness of samples limits penetration of the light emitted by the microscope, whereas the opacity of the nervous tissue due to its high lipid content limits the resolution of objects, making the acquisition of images difficult. The CLARITY (Clear, Lipidexchanged, Acrylamidehybridized Rigid, Imaging/Immunostaining/In situ hybridizationcompatible, Tissue hYdrogel) technique makes possible to remedy these disadvantages. This technique was adapted in our laboratory for the study of the rat spinal cord. According to the described procedure we obtained a completely translucid and structurally intact organ that allowed processing through immunofluorescence and lectinhistochemistry techniques without major drawbacks. Confocal images of higher resolution and greater penetrability in comparison with those captured using conventional
processing techniques were obtained from more than 1 mm thick samples. The implementation of this technique in our laboratory will improve the information obtained from our samples of interest.