Qwin v3

Bronchoscopy was performed 6 h after exposure with a flexible video bronchoscope (EB-580S, FUJIFILM Corporation, Japan). Topical anaesthesia with lidocaine was applied to the pharynx and bronchial tree. Bronchial wash (BW) 2 × 20 ml and Bronchoalveolar lavage (BAL) 3 × 60 ml with sterile saline were performed. Aspirates from BW and BAL were placed on ice in separate siliconized containers. Lavage samples were filtered through a nylon filter (100 µm) and centrifuged at 400 × g for 15 min. Cell pellets were resuspended in 0.9% NaCl at a concentration of 10⁶ cells/mL. Differential cell counts were performed on cytocentrifuge preparations stained with May-Grünwald Giemsa. 400 cells per slide were counted. Images were taken with LeicaQWin V3 (Leica Q500IW; Leica, Cambridge, UK).

Isolated SC were seeded on cover slides (ᴓ12 mm, Menzel) in a Primaria-treated 24-well plate (VWR International) in which wells were coated with Collagen A (0.5 mg/ml, Biochrom). After cultivation in growth medium, cells were fixed with formaldehyde (3.7%) in PBS. Cells were permeabilised in PBS containing Triton X-100 (0.5%) for 20 min. Cells were then blocked in PBS with 20% normal serum and 0.5% Triton X-100 for 1 h. Incubation with primary antibody was performed overnight. Mouse anti-Pax7 antibody (Developmental Studies Hybridoma Bank) was diluted 1:50 in normal serum (2%) and TritionX-100 (0.5%) in PBS. After being washed with PBS, samples were incubated with a rabbit anti-mouse Alexa488 antibody (Thermo Fisher Scientific, 1:1000) for 1 h at room temperature, and subsequently, cell nuclei were stained with DAPI. For fluorescence microscopy, the Leica DM4000B and its associated software Leica QWin V3 were used. Micrographs were merged by using Adobe Photoshop CS5; contrast and brightness were adjusted to the same degree in every sample group.

The histological bladess were stained with hematoxylin and eosin (Merk & Co., Inc., Kenilworth, New Jersey, NJ, USA). The inflammatory response was determined through qualitative analysis in which all histological slices did not show any different cells related to the tissue response, with more emphasis in lymphocytes.
One histological blade from each animal for the experimental time was chosen and two sections photographed under an image processing system, which consisted of a light microscope (DM 4000B, Leica), a color image processor (Leica Qwin V3, Leica software), a color camera (DFC 500, Leica), and a computer (Intel Core I5, intel Corp, Santa Clara, CA, USA; Windows 10, Microsoft Corp, Redmond, DC, USA) connected with ImageJ digitized image analyzer software (National Institutes of Health, Bethesda, MD, USA).
All samples had their identification hidden, so the examiner was blind. Three regions were assessed: the first image was taken in the center of the defect, followed by the one on the right and one on the left, totaling 72 images per experimental time/per group, at a ×100 magnification. All images were imported to the ImageJ software using a grid tool with 130 points that allowed enough points to perform the cell counting. Therefore, all points that crossed those cells were counted as numbers of cells to compare among groups.