The diverse composition of mammalian tissues poses challenges for understanding the cell–cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only two to six molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a method that overcomes major impediments to highly multiplex tissue imaging. “Iterative bleaching extends multiplexity” (IBEX) uses an iterative staining and chemical bleaching method to enable high-resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an “open” system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, cellular indexing of transcriptomes and epitopes by sequencing, and IBEX analysis of the same tissue. To facilitate data processing, we provide an open-source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high-content imaging to reveal the complex cellular landscape of diverse organs and tissues.
High content imaging is needed to catalogue the variety of cellular phenotypes and multi-cellular ecosystems present in metazoan tissues. We recently developed Iterative Bleaching Extends multi-pleXity (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 over 250 commercially available antibodies, 16 unique fluorophores, and can be easily adopted to different imaging platforms using slides and non-proprietary imaging chambers. The overall protocol consists of iterative cycles of antibody labelling, imaging, and chemical bleaching that can be completed at relatively low cost in 2-5 days 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 an open and versatile 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.
Purpose of review In this era of modern combination antiretroviral therapy (cART) HIV-associated neurocognitive disorders (HAND) continues to be a debilitating condition affecting a large portion of the infected population. In this review we highlight recent discoveries that help to define the interplay between HIV life cycle, the innate immune system, and cellular autophagy in the context of the CNS. Recent findings Investigators have recently elucidated themes in HAND, which place it in a unique framework. Cells of macrophage lineage and probably astrocytes play a role in disseminating virus through the CNS. Each of these cell types responds to a diverse population of constantly evolving virus existing in an inflammatory environment. This occurs though the failure of both host antiviral mechanisms, such as autophagy, and innate immunological signaling pathways to control viral replication. Summary The newest findings detailed in this review help define why HIV CNS disease is a difficult target for therapeutics and create hope that these new mechanisms may be exploited to attenuate viral replication and eliminate disease.
Induction of the kynurenine pathway (KP) of tryptophan (TRP) catabolism has been proposed to contribute to T cell dysfunction during human/simian immunodeficiency virus (SIV) infection via depletion of local TRP levels and production of immunomodulatory KP metabolites. However, while changes in TRP and KP metabolites have been observed in plasma, their levels in lymphoid tissues and levels of enzymes downstream of indoleamine 2,3-dioxygenase (IDO1) have been relatively unexplored. We used our SIV-infected pigtailed macaque model to analyze longitudinal changes in KP metabolites and enzymes by gas chromatography/mass spectrometry and NanoString nCounter gene expression analysis, respectively, in spleen and blood compared to changes previously established in brain and CSF. We found that TRP levels were remarkably stable in tissue sites despite robust depletion in the circulating plasma and CSF. We also demonstrated that intracellular TRP reserves were maintained in cultured cells even in the presence of depleted extracellular TRP levels. Kynurenine (KYN), 3-hydroxykynurenine, quinolinic acid, and the KP enzymes all displayed highly divergent patterns in the sites examined, though IDO1 expression always correlated with local KYN/TRP ratios. Finally, we demonstrated by fluorescence-activated cell sorting that myeloid dendritic cells and cells of monocytic lineage were the highest producers of IDO1 in chronically infected spleens. Overall, our study reveals insights into the tissue-specific regulation of KP enzymes and metabolites and, in particular, highlights the multiple mechanisms by which cells and tissues seek to prevent TRP starvation during inflammation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.