Simple Yet Versatile Fluorescent Labeling Methods for Advanced Microscopy Techniques

Abstract

Advanced microscopy techniques have undergone rapid development in recent years and now enable the comprehensive 3-dimensional analysis of intact biological specimens over a wide range of length scales. Despite these advances, the technologies for labeling specimens and which are critical to their success have developed at a slower pace. Immunofluorescence and fluorescent protein tags that have been used for decades are still the two dominant methods in use today. Although powerful, they still face several limitations such as slow labeling speed due to the antibody’s large size (particularly for thick specimens), inconsistent reproducibility due to antibody variability, and complicated processing due to the required genetic manipulation of the specimen (if possible) when using fluorescent proteins. In addition, the labeling efficiency is challenging for both approaches because many antibodies are unable to bind heavily fixed or processed specimens, and fluorescent protein can dimerize or otherwise perturb the normal function of the organism and may become nonfluorescent due to denaturation and/or dissociated during potentially harsh sample processing conditions. Here, I present a new labeling method that is inspired by well-established histological staining. The new labeling method uses commercially available reagents to efficiently map the distributions of proteins and oxidized carbohydrates of various specimens, so it is simple, versatile, and easily accessible. I also demonstrate its powerful utility when combining with advanced microscopy techniques and/or immunofluorescence. Additionally, I provide step-by-step procedures for a range of specimens and address ways to customize and troubleshoot to help disseminate this method.