Protocols

As part of our commitment to excellence we strive to make research easily reproducible. We encourage the publication of detailed protocols covering all aspects of the experimental work including hardware construction, sample preparation, image settings, and computational analysis of the final images. Ideally, all protocols would be hosted on a single platform (e.g. protocols.io).

While that is the ideal, in practice we know there are multiple protocol repositories and we do not want to limit your choices. This page serves as a central location, referencing protocols across the various hosting platforms.

Have a protocol that you would like to share? Follow the how to contribute instructions.

Title	Details
IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues [detailed protocol].	High-content imaging is... 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.
EdU Immunohistochemistry using Click-it reaction [detailed protocol].	This protocol allows... 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.

Title

Details

IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues [detailed protocol].

High-content imaging is...

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.

EdU Immunohistochemistry using Click-it reaction [detailed protocol].

This protocol allows...

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.