High-content imaging is needed to catalog the variety of cellular phenotypes and multicellular ecosystems present in metazoan tissues. We recently developed iterative bleaching extends multiplexity (IBEX), an iterative immunolabeling and chemical bleaching method that enables multiplexed imaging (>65 parameters) in diverse tissues, including human organs relevant for international consortia efforts. IBEX is compatible with >250 commercially available antibodies and 16 unique fluorophores, and can be easily adopted to different imaging platforms using slides and nonproprietary imaging chambers. The overall protocol consists of iterative cycles of antibody labeling, imaging and chemical bleaching that can be completed at relatively low cost in 2-5 d by biologists with basic laboratory skills. To support widespread adoption, we provide extensive details on tissue processing, curated lists of validated antibodies and tissue-specific panels for multiplex imaging. Furthermore, instructions are included on how to automate the method using competitively priced instruments and reagents. Finally, we present a software solution for image alignment that can be executed by individuals without programming experience using open-source software and freeware. In summary, IBEX is a noncommercial method that can be readily implemented by academic laboratories and scaled to achieve high-content mapping of diverse tissues in support of a Human Reference Atlas or other such applications.

This protocol allows users to image histology tissue using basic immunohistochemistry techniques in combination with EdU capture (using the Click-it reaction system). EdU (5-ethynyl-2'-deoxyuridine) will capture S-phase cells with the thymidine analogue, allowing to take a snap-shot of actively diving cells. In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor dye in a fast, highly-specific click reaction. This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol.

This is a step-wise protocol for making chrome alum gelatin adhesive and coating microscopy slides for better tissue adherence. This protocol is useful for researchers who are unable to purchase Chrome Alum-Gelatin Adhesive (Newcomer Supply catalog #1033A) in their country.

The goal of this protocol is to evaluate the optimal snap (flash) freezing method for multiplexed tissue imaging. We detail four different methods (see sample preparation) for preparing snap (flash) frozen tissues. We additionally compare the image quality, ease of use, and safety of each method to our preferred method of tissue preservation (fixed frozen with sucrose cryopreservation). To directly compare tissue preservation methods, tissues were collected from one mouse and divided among the five groups. We determined that the optimal protocol for snap frozen tissues is cryogenic freezing with the Seal'N Freeze Box followed by post-fixation of sections using 1% paraformaldehyde for 10 minutes at room temperature. This method was easy to do, froze the tissue quickly without tissue artifacts, and allowed for immunolabeling of antibodies after fixation.