Abstract

One of the challenges in labeling tissues for fluorescence microscopy is minimizing sample processing while maintaining or improving the information generated by the fluorescent label. Generally, tissues are extracted, fixed, and embedded in mounting media (such as paraffin), sectioned, and then postprocessed by removing the paraffin, blocking, labeling, and washing. Despite all of these steps, the consistency of labeling quality can vary as a result of several factors, including heterogeneity in labeling efficiency from slide to slide, the necessity of postprocessing to obtain information on sequential sections of tissue, interference from the mounting media, and loss of native three-dimensional structural information, especially in thicker sections. A method for embedding and processing tissues that have been labeled by intravital staining is described in this study. Intravital staining is the process in which live-cell dyes and other labels are injected into the bloodstream before fixation of the tissues. Tissues processed this way can be imaged upon sectioning without further staining and retain their native, three-dimensional information, thereby improving the information retained by the labels and speeding up sample processing.