Infected neutrophils leave the skin via afferent lymphatics after BCG injection and shuttle fluorescent bacilli to the ADLNs. (A) A total of 106 CFUs of rBCG-dsred and rBCG-egfp fluorescent strains was injected into 2 adjacent distinct sites of the ear dorsum (left panel). Four hours later, ADLN cryosections were laser scanned under a confocal microscope to colocalize red or green bacilli with Ly-6G+ cells (blue). Neutrophils carrying either red or green bacilli or coinfected with both strains in the ADLNs were scored in 7 fields from 2 ADLN sections. Right panel is control coinjection of mixed red and green bacilli in the same site. Cryosections were labeled with Alexa fluor 633 (blue) mounted in Fluoromount. Otherwise, images were acquired as in Figure 4Diii. (B) Four hours after BCG-egfp inoculation, ear sections were immunostained with anti-Lyve-1 (brown) and neutrophils were detected either by counterstaining polylobed nuclei with hematoxylin or by anti-Ly-6G (blue). Neutrophils were detected inside the lumen of lymphatic vessels in the injection site vicinity. After antibody treatment, paraffin embedded sections were preserved in Aquamount and observed under a Nikon Microphot FXA light microscope with Plan Apo 60 ×/1.40 NA oil iris objective (left) or 40 ×/0.70 NA objective (right). Images were acquired with a Nikon DX digital camera and processed with the Nikon capture software. (C) Four hours following BCG-egfp inoculation, ear dermis was stained with anti-Lyve-1 (red) and anti-Ly-6G (blue), and a 3-dimensional “orthogonal” slice projection was analyzed by confocal microscopy. The large central panel shows a single image among 46 slices recorded at 0.23-μm intervals. To characterize cells inside lymphatic vessels (underlined by red dashes), the x-axis (green line) and y-axis (red line) were defined for sliced z-axis reconstruction. The corresponding results for the x, z slice and y, z slice are shown and the crossing point between green and red lines represents the z-stack position of the central panel image. A neutrophil carrying bacilli inside the lymphatic vessel lumen is depicted. Dermis cryosections were labeled with Alexa fluor 594 (red) and 633 (blue) mounted in Fluoromount under a Zeiss Axioskop 2FS with a Plan-APOCHROMAT 63 ×/1.4 NA objective and zoomed 2.9 ×. Images were acquired and processed with Zeiss LSM 510 software.
