Strangward, P. | bioRxiv Science

bioRxiv2025

RatDISCO, a tissue clearing and immunolabelling protocol for large rat brains.

RatDISCO is a simple, cost-effective, and reproducible tissue-clearing protocol optimised for immunolabeling in adult rat brains. It enables robust detection of diverse neuronal subtypes, glial populations, and vasculature, overcoming key limitations in antibody penetration and optical transparency. It is compatible with virally labelled-and transgenic mice, as well as human-derived brain organoids, highlighting its versatility across species and models. Functionally, it enables the detection of activity-dependent markers after behaviour, verified in a rat model of neurodevelopmental disorders, the Fragile X syndrome. These findings highlight RatDISCO as a broadly applicable tool for investigating neuroanatomical and functional alterations in rat models of disorders. HighlightsO_LIOptimised for immunolabeling in adult rat brains C_LIO_LIEnables detection of neurons, glial cells, and vasculature C_LIO_LIAchieves high tissue transparency without requiring specialised equipment. C_LIO_LICompatible with virally labelled mice and human brain organoids C_LIO_LIFacilitates mapping of activity-dependent markers in rat models of neurodevelopmental disorders C_LI

neuroscience↗

bioRxiv2023

CD163 and Tim-4 identify resident intestinal macrophages across sub-tissular regions that are spatially regulated by TGF-β

In bodily organs, macrophages are localised in poorly understood tissular and sub-tissular niches associated with defined macrophage ontogeny and activity. In the intestine, a paradigm is emerging that long-lived macrophages are dominantly present in the muscular layer, while highly monocyte-replenished populations are found in the lamina propria beneath the epithelial barrier. Whether longevity is restricted in such a simplified manner has not been well explored. Moreover, the impact of specific gut-associated factors on long-lived macrophage functionality and niche occupancy is unknown. We generated sc-RNA-Seq data from wild-type and Ccr2-/- mice to identify phenotypic features of long-lived macrophage populations in distinct intestinal niches and identified CD163 as a useful marker to distinguish submucosal/muscularis (S/M) from lamina propria (LP) macrophages. Challenging the emerging paradigm, long-lived macrophages, identified by Tim-4 expression, were found in the LP and S/M. Long-lived LP macrophages are restrained in their response to proinflammatory stimulation compared to short-lived populations in the same location, and to the long-lived population within the S/M. Employing a novel Timd4creTgfbr2fl/fl mouse line we demonstrate distinct functions of TGF-{beta} on long-lived macrophages in these two compartments. Importantly, in Timd4creTgfbr2fl/fl mice, zonation of CD163+ macrophages in the S/M was lost, suggesting TGF-{beta} plays an unappreciated role in positioning of macrophages in the tissue. These data highlight the importance of considering ontogeny and niche when assessing the action of key intestinal regulatory signals.

immunology↗