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Our protocol describes a simple procedure for imaging thick lymph node sections by 2-photon microscopy. Lymph nodes are sectioned using a vibratome (vibrating microtome) to produce slices of tissue that can then be stained with fluorescently labeled antibodies. The thick tissue sections (150-200 μm depth) allow for the detection of cell clustering that is typically under-represented in thin sections (10-20 μm) used for conventional confocal microscopy. Application of 2-photon microscopy facilitates imaging through the thick volume of the vibratome sections. In combination with automated image processing software, a thick lymph node cross-section image also facilitates quantitation of cellular events within a relatively large area of the tissue, thus providing a clearer picture on the spatial distribution of cellular events of interest (e.g., T cell clustering). This method can also readily be applied to other tissues, such as the spleen or skin.