Tcs sp8 upright microscope

Embryos were anaesthetised in 0.014% tricaine (MS-222, Sigma-Aldrich), mounted in a 35 mm glass-bottomed petri dish (0.17 mm, MatTek) using 0.6-1% low melting point agarose (Sigma-Aldrich) containing 0.014% tricaine, and bathed in Danieau's buffer containing 0.007 (0.5×) to 0.014% (1×) tricaine and 0.003% PTU (as indicated). Time-lapse imaging was performed using a Leica TCS SP8 upright microscope with a Leica HCX IRAPO L ×25/0.95 water-dipping objective and heating chamber, or on an upright 3i spinning-disc confocal using a Zeiss Plan-Apochromat 20×, 40× or 63×/1.0 NA water-dipping objective. Image processing was performed using Fiji software (Schindelin et al., 2012 (link)).

Whole-mount brains were mounted on a 76 × 26 mm glass microscope slide (KnittelGlass) to which pairs of Paper Reinforcement Rings had been applied. The samples were mounted in Prolong Gold mounting solution (Invitrogen). Spacers were covered with a cover glass (KnittelGlass, #1 thickness, 0.17 mm) held in place by nail polish. Image acquisition was done sequentially on a Leica TCS SP8 upright microscope with a 25 × 0.95 NA plan-apochromat water immersion objective. The original image data consisted of 1, 024 × 1, 024 × ~200 voxels, with a voxel size of 0.60 × 0.60 × 0.98 μm. Images were acquired with a 12-bit dynamic range. A frame average of two successive scans was applied. Fluorescence emission from the 488, 547, and 647 nm was imaged using the 488, 561, and 633 nm lasers, respectively. The laser power was increased along the z-axis to compensate for signal attenuation.

LysM^GFP mice were injected in the right footpad with 2.5 × 10⁷ CFU S. aureus. An hour later mice received intravenously injected Cell Tracker Red CMTPX (Thermofisher) labeled lymphocytes. An hour after lymphocyte injection mice were injected intravenously with a mixture of anti-PNAd⁻Dylight594 and albumin-Dylight-680 to visualize HEV and vasculature, respectively. Two-photon videomicroscopy was employed to assess the movement and location of neutrophils and lymphocytes in lymph node blood vessels following infection with S. aureus. The right hindlimb popliteal LN was exposed for imaging in the anesthetized mouse. The LN was imaged from 2 to 4 hpi. Image acquisition was performed an upright two-photon microscope (Leica Biosystems TCS SP8 Upright Microscope). For antibody blocking studies, mice were pretreated with blocking antibody 20 min before infection.