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Published on: April 8, 2015
Protocol for isotype-resolved 3D imaging and quantification of bone marrow plasma cells in murine femurs
Abdouramane Camara1, Baizhi Chen2, An Qi Xu2
1Immunity and Cancer Laboratory, The Francis Crick Institute, London, UK; West African Centre for Cell Biology of Infectious Pathogens, University of Ghana, Accra, Ghana.
Bone marrow plasma cells maintain durable antibody responses, but their tissue localization and morphology are difficult to quantify in situ. Here, we present a protocol for isotype-resolved 3D imaging and quantification of bone marrow plasma cells in murine femurs. We describe steps for fixation, optimal cutting temperature (OCT) coumpound embedding, longitudinal opening of mouse femurs, whole-mount immunostaining, optical clearing, and confocal 2D/3D imaging. We detail an Imaris-based workflow for reproducible single-cell segmentation, isotype-resolved counting, and the extraction of morphological features from defined regions of interest.
Bone marrow plasma cells maintain durable antibody responses, but their tissue localization and morphology are difficult to quantify in situ. Here, we present a protocol for isotype-resolved 3D imaging and quantification of bone marrow plasma cells in murine femurs. We describe steps for fixation, optimal cutting temperature (OCT) coumpound embedding, longitudinal opening of mouse femurs, whole-mount immunostaining, optical clearing, and confocal 2D/3D imaging. We detail an Imaris-based workflow for reproducible single-cell segmentation, isotype-resolved counting, and the extraction of morphological features from defined regions of interest.
