Digital camera

Lungs explanted from 7–9 week human fetal tissue and E12.5 mice were cultured for 48–72 hours according to previous published protocols10 (link)27 (link)28 (link)29 (link). Human fetal lungs were separated into two halves with the trachea alternatively kept with the left or right lung in the same experimental series, a manoeuvre which did not affect lung growth. Timelapse images were captured at 0, 24, 48 and 72 hours with a dissecting microscope equipped with a digital camera (Leica Microsystems, Milton Keynes, U.K.).
For the fetal Ca²⁺_o conditions, [Ca²⁺]_o in the DMEM-F12 medium was increased from 1.05 mM [Ca²⁺]_o to 1.70 mM [Ca²⁺]_o using 1 M CaCl₂ (Sigma-Aldrich, Gillingham, U.K.). Inhibitors of chloride-transporting mechanisms included the wide-spectrum anion exchange inhibitor 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) (Sigma-Aldrich) and the CFTR specific inhibitor Inh-172 (Tocris) and the loop diuretic, bumetanide (Sigma-Aldrich). All chloride channel inhibitors were dissolved in DMSO, which has previously been shown not to affect lung explant growth10 (link). Vehicle control experiments were performed by adding the equivalent amount of DMSO to the lung cultures.

MoMΦ were cultured in 4-well chamber slides (3 × 10⁵ live cells per well), stimulated with IL-10 or TGF-β or left untreated. Then, 24 h post-stimulation, cells were fixed with 4% paraformaldehyde, washed with PBS, and subsequently phase-contrast microscopy images were acquired using an inverted stereo microscope (Olympus IX 70, Segrate, Italy). The morphology of untreated and treated samples was further evaluated though May–Grunwald–Giemsa (MGG) staining: fixed cells were stained using the MGG method, and digital computer images were recorded at 20× using a light microscope coupled with a digital camera (Leica Microsystem, Welzlar, Germany) [42 (link)].

Retinal sections (10-μm thick) were fixed in 4 % paraformaldehyde, and incubated with rhodamine-conjugated peanut agglutinin (Vector Laboratories; Burlingame, CA, USA) at room temperature for 1 h. Alternatively, they were incubated with rabbit anti-GFAP antibody (1:500; DAKO, Carpinteria, CA, USA) overnight at 4 °C, followed by incubation with Alexa 488-conjugated goat anti-rabbit IgG at room temperature for 1 h. All the sections were examined under a microscope equipped with a digital camera (Leica Microsystems, Wetzlar, Germany). Four mice for each group were used to prepare the retinal sections.

Specimen pictures, taken with a digital camera (Leica Microsystems, Wetzlar, Germany) were analyzed using ImageJ software [45 (link)]. At a magnification of 5×, the percentage antibody positivity for COL I, COL III, and HABP were obtained. At least five pictures from five sections for every specimen were counted. Data obtained from the analysed images were averaged to obtain the representative values. Results were expressed as percentage antibody positivity per unit area (where the unit area was equivalent to the field covered at 5× magnification). The image analysis was made by a single reader in different times. We calculated the intra-reader reliability by Intra-class-correlation coefficient, ICC_2,k: 0.9 (0.85–0.95).

CEA specimens were processed as described in detail previously [7 (link)]. Briefly, immunohistochemistry of 4 μm paraffin sections followed the alkaline phosphatase method [7 (link)]. For immunohistochemical detection of macrophages (CD68), T-cells (CD3), and neovascularization (CD31), specimens were stained automatically using the Ventana Discovery XT staining system (Ventana, Tucson, USA). CD31 is a transmenbrane glycoprotein that is mainly expressed by endothelial cells. Thus, CD31 labels neovascularization within carotid plaques [22 ]. Additional stains with van Gieson’s and hematoxylin/eosin were performed for verification and spatial classification of immunohistochemical results. For quantitative evaluation, the StereoInvestigator system (Micro Brightfield Europe, Magdeburg, Germany) was used in combination with a spectral confocal microscope and a digital camera (Leica Microsystems, Heidelberg, Germany). As macrophages tend to appear as confluent infiltrates (Fig 3), the total area occupied by macrophages was measured. The density of CD31-positive vessels and the density of T cells (Fig 3) were determined by counting the cells in relation to the total section area (n/mm²). The extent of macrophage infiltration was quantified by calculating the percentage of the area occupied by macrophages relative to the total section area.

The lung tissues were collected and infused with 4% paraformaldehyde and were then dehydrated, paraffin-embedded, and cut into 5-µm-thick sections. The sections were then stained with hematoxylin for 3 min and eosin for 3 min. Finally, the pathological morphology of the mouse lung tissue was observed using a digital camera (Leica Microsystems Inc., IL, USA).