Abstract
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.
Keywords: 2-photon microscopy, Lymph nodes, T lymphocytes, Vibratome, Immune response
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 6. Example image of LN slice acquired using 2-photon microscopy. Shown is a 150 μm slice of mouse inguinal LN populated by CellTrace Violet-labelled CD4 T cells (green), CellTracker Deep Red-labelled CD8 T cells (white) and TRITC-labelled migratory DC (red). Cells were labelled with CellTrace or CellTracker dyes as per the manufacturer protocols (Thermo Fisher Scientific). LN sections were then stained with Pacific Blue anti-B220 (blue). Also in blue was collagen delineating the LN capsule illuminated via second-harmonic generation. Image was acquired using Zeiss LSM710 NLO at dual wavelengths (800 nm for excitation of CellTrace Violet, CellTracker Deep Red and Pacific Blue, and 880 nm for TRITC excitation) and at 512 x 512 resolution in 8 x 4 tiles for a 3 x 1.5 mm LN cross-section. Image was then processed using Imaris (Bitplane). Autofluorescence and spectral crossover were removed using Channel Arithmetic function in Imaris XTension.
Notes
Recipes
Acknowledgments
This protocol was adapted from our publication (Hor et al., 2015). This work was supported by the National Health and Medical Research Council, and by the Australian Research Council.
References
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