Dataset Information

Whole-mount immunofluorescence imaging and isolation of mesothelium-bound immune cell aggregates during mouse peritoneal inflammation.

ABSTRACT: Resident peritoneal macrophages (resMØs) are crucial for repairing peritoneal injuries and controlling infections by forming mesothelium-bound resMØ-aggregates in the peritoneal wall and omentum. Here we present a protocol to analyze these structures in mouse models of peritoneal inflammation. We describe the dissection, fixation, immunofluorescent staining, and mounting of whole peritoneal wall and omentum samples and subsequent confocal microscopy imaging of resMØ-aggregates. We also detail the steps to isolate resMØ-aggregates for additional studies, including flow cytometry and electron-microscopy-based analysis. For complete details on the use and execution of this protocol, please refer to Vega-Pérez et al. (2021).¹.

SUBMITTER: Ferriz M

PROVIDER: S-EPMC9898286 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Whole-mount immunofluorescence imaging and isolation of mesothelium-bound immune cell aggregates during mouse peritoneal inflammation.

Ferriz Margarita M Vega-Pérez Adrián A Gutiérrez-González Alejandra A Alvarez-Ladrón Natalia N Ardavín Carlos C

STAR protocols 20230129 1

Resident peritoneal macrophages (resMØs) are crucial for repairing peritoneal injuries and controlling infections by forming mesothelium-bound resMØ-aggregates in the peritoneal wall and omentum. Here we present a protocol to analyze these structures in mouse models of peritoneal inflammation. We describe the dissection, fixation, immunofluorescent staining, and mounting of whole peritoneal wall and omentum samples and subsequent confocal microscopy imaging of resMØ-aggregates. We also detail th ...[more]

PMID: 36825810

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Project description:Multiplex immunohistochemistry/immunofluorescence (mIHC/mIF) is a developing technology that facilitates the evaluation of multiple, simultaneous protein expressions at single-cell resolution while preserving tissue architecture. These approaches have shown great potential for biomarker discovery, yet many challenges remain. Importantly, streamlined cross-registration of multiplex immunofluorescence images with additional imaging modalities and immunohistochemistry (IHC) can help increase the plex and/or improve the quality of the data generated by potentiating downstream processes such as cell segmentation. To address this problem, a fully automated process was designed to perform a hierarchical, parallelizable, and deformable registration of multiplexed digital whole-slide images (WSIs). We generalized the calculation of mutual information as a registration criterion to an arbitrary number of dimensions, making it well suited for multiplexed imaging. We also used the self-information of a given IF channel as a criterion to select the optimal channels to use for registration. Additionally, as precise labeling of cellular membranes in situ is essential for robust cell segmentation, a pan-membrane immunohistochemical staining method was developed for incorporation into mIF panels or for use as an IHC followed by cross-registration. In this study, we demonstrate this process by registering whole-slide 6-plex/7-color mIF images with whole-slide brightfield mIHC images, including a CD3 and a pan-membrane stain. Our algorithm, WSI, mutual information registration (WSIMIR), performed highly accurate registration allowing the retrospective generation of an 8-plex/9-color, WSI, and outperformed 2 alternative automated methods for cross-registration by Jaccard index and Dice similarity coefficient (WSIMIR vs automated WARPY, P < .01 and P < .01, respectively, vs HALO + transformix, P = .083 and P = .049, respectively). Furthermore, the addition of a pan-membrane IHC stain cross-registered to an mIF panel facilitated improved automated cell segmentation across mIF WSIs, as measured by significantly increased correct detections, Jaccard index (0.78 vs 0.65), and Dice similarity coefficient (0.88 vs 0.79).

Dataset Information

Whole-mount immunofluorescence imaging and isolation of mesothelium-bound immune cell aggregates during mouse peritoneal inflammation.

Publications

Whole-mount immunofluorescence imaging and isolation of mesothelium-bound immune cell aggregates during mouse peritoneal inflammation.

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