An innovative strategy to obtain extraordinary specificity in immunofluorescent labeling and optical super resolution imaging of microtubules

Abstract

When performing immunofluorescent labeling of microtubules, Triton X-100 (TX100) is commonly used as the cell membrane permeabilization agent to improve the accessibility of antigens. Usually, before immunofluorescent labeling, cells are fixed first by aldehydes, followed by permeabilization with TX100. Here, we report an innovative immunofluorescent labeling strategy for microtubules with a meaningful alteration, that is, to treat cells with TX100 first and fix with aldehydes later. We proved that this subtle change can greatly improve the specificity of microtubular immunolabeling. However, treating cells first with TX100 can also severely disrupt the integrity of microtubules if an excessive amount of TX100 is used. Hence, TX100 is a “double-edged sword” in immunofluorescent labeling of microtubules and elaborative control of its dosage is required. In the experiment, we compared different immunofluorescent labeling protocols using various cell lines and found that treating cells with 0.02% TX100 before fixation is an optimal solution. Confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM) and single molecule localization microscopy (SMLM) are utilized to verify the immunofluorescent labeling results performed via the presented unusual protocol. It is possible that such a modified immunofluorescent labeling protocol of microtubules can be generalized as a universal strategy.