Advances in light-sheet and confocal microscopy now allow imaging of cleared large biological tissue samples and enable the three-dimensional appreciation of cell and protein localization in their native organ environment. However, the sample 2 preparations for such imaging are often onerous and their capability for antigen detection limited. Here we describe FLASH (Fast Light-microscopic analysis of Antibody-Stained wHole organs), a simple and rapid, fully customizable technique for molecular phenotyping of intact tissue volumes. FLASH utilizes non-degradative epitope recovery and membrane solubilisation to enable the detection of a multitude of membranous, cytoplasmic and nuclear antigens in whole mouse organs and embryos, human biopsies, organoids and Drosophila. Retrieval and immunolabelling of epithelial markers, an obstacle for previous clearing techniques, can be achieved with FLASH. Upon volumetric imaging, FLASH-processed samples preserve their architecture and integrity, and can be paraffin-embedded for subsequent histopathological analysis. The technique can be performed by scientists trained in light microscopy and yields results in less than one week.
Reproduction induces increased food intake across females of many animal species 1 – 4 , providing a physiologically relevant paradigm for exploration of appetite regulation. Parsing enteric neuronal diversity in Drosophila , we identify a key role for gut-innervating neurons with sex- and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, leading to post-mating release of their neuropeptide onto the muscles of the crop: a stomach-like organ. Post-mating neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake. Preventing enteric neuron remodelling blunts reproductive hyperphagia and reduces reproductive fitness. Thus, plasticity of enteric neurons is key to reproductive success. Our findings provide a new mechanism to attain the positive energy balance that sustains gestation which, if dysregulated, could contribute to infertility or weight gain.
Animal behavior is encoded in neuronal circuits in the brain. To elucidate the function of these circuits, it is necessary to identify, record from and manipulate networks of connected neurons. Here we present BAcTrace ( B otulinum Ac tivated Tracer ), a genetically encoded, retro-grade, transsynaptic labelling system. BAcTrace is based on C. botulinum neurotoxin A, Botox, which we have engineered to travel retrogradely between neurons to activate an otherwise silent transcription factor. We validate BAcTrace at three neuronal connections in the Drosophila olfactory system. We show that BAcTrace-mediated labeling allows electrophysiological recordings of connected neurons. Finally, in a challenging circuit with highly divergent connections, BAcTrace correctly identifies 12 out of 16 connections, which were previously observed by electron microscopy.
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.