Caseviewer 3

The left lung was removed, fixed in Milloning buffer solution (pH 7.4) with 4% paraformaldehyde to preserve pulmonary architecture. Briefly, samples were embedded in paraffin (Sigma-Aldrich), and 4 μm-thick sections were cut and stained with hematoxylin and eosin for quantification of granuloma area, Picrosirius for collagen fibers and Sirius Red (pH 10.2) for neutrophils and eosinophils counted in the parenchyma and in peribronchiolar area, respectively. Slides were scanned with 3DHISTECH–Pannoramic MIDI whole slide scanner (capture with a 20× objective lens) and the resulting images analyzed with CaseViewer 3.3, Pannoramic Viewer 1.15.4, and HistoQuant softwares (3DHISTECH). Silica crystals were analyzed, in 15 independent fields, with a light microscope (Olympus BX50) equipped with polarizing attachment for detecting birefringent particles and Image-Pro Plus Version 4.

Left lungs from each experimental group were dissected and placed in Millonig fixative solution (pH = 7.4) with 4% paraformaldehyde for 48 h to preserve tissue architecture. Tissues were dehydrated and clarified in xylene before paraffin embedding. Lung sections of 4 μm were stained with hematoxylin and eosin (H&E), to analyze granulomas, or Picrosirius, to evaluate collagen deposition, in light microscope using 3DHISTECH–Pannoramic MIDI whole slide scanner (capture with a 20× objective lens) and the resulting images analyzed with CaseViewer 3.3, Pannoramic Viewer 1.15.4, and HistoQuant softwares (3DHISTECH). Silica crystals were analyzed, in 15 independent fields, with a light microscope (Olympus BX51) equipped with polarizing attachment for detecting birefringent particles and the quantification were performed using the software Image-Pro Plus Version 6.2 (Media Cybernetics Inc., Rockville, MD, United States).