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Additional Lignocaine 1% solution was instilled into lung segments to dampen the cough reflex. The bronchial tree was inspected by autofluorescence bronchoscopy (SAFE 3000; Pentax, Tokyo, Japan). Two endobronchial biopsies per patient were taken at B4-6 of the right lung with a cup forceps (Pentax KW2411S). selleck products Special care was taken in positioning the forceps laterally to the bronchial carina to minimize the amount of connective tissue and at the same time to maximize the yield of ASM in the biopsies. After collection, the biopsy specimens were fixed in 4% buffered formaldehyde and embedded in paraffin. Biopsy specimens were cut into 4-��m sections and stained with H&E for initial analysis as described previously [7, 9]. Next, sections were stained with Elastica-van Gieson for analysis of elastin in the ASM layer. Antigen retrieval and the primary antibodies used for labelling of ECM (collagen I, III and IV, decorin, versican, fibronectin, laminin and tenascin) are shown in Table?1. Briefly, the paraffin sections were dewaxed and rehydrated. Prior to overnight incubation with the primary antibody, a 3% SCH772984 clinical trial H2O2 solution was applied for 40?min to inhibit endogenous peroxidase activity. A streptavidin-biotin (LSAB kit; DAKO, Glostrup, Denmark) or a nonbiotin (Novocastra Novolink; Leica Biosystems, Newcastle Upon Tyne, UK) detection method was applied as secondary antibody staining. All sections were incubated with antibodies from the same batch within one session and counterstained with Harris haematoxylin. Negative control staining was performed by replacing the primary antibodies by phosphate-buffered saline (PBS) or isotype-matched control antibodies. Images of the microscopic slides were captured digitally (Leica DMR; Leica Microsystems, Wetzlar, Germany). Selection of the ASM area for analysis was based on alpha-smooth muscle actin (SMA) stain and morphology of the ASM cells, which were elongated and disposed Ritonavir in bundles [18]. The area of positive staining for each antibody within the selected ASM was determined by colour threshold. To define this threshold, sections of 6�C8 subjects per study group stained with each antibody were analysed to achieve the best range of positivity. Afterwards, the colour data file created for each antibody was applied to all cases stained with the same antibody using Image-Pro Plus 4.1 (Media Cybernetics, Bethesda, USA) at 200�� magnification as described previously [9]. Fractional area was expressed as percentage of the total selected ASM area. Mean density of the immunohistochemical staining was measured by the image analysis software. Fractional area and mean density were compared between study groups using unpaired t-test or Mann�CWhitney U-test. Correlations between ECM and airway function parameters were examined using Pearson's or Spearman's correlation coefficient. Statistical analyses were performed using SPSS 18 (IBM Corporation, Armonk, NY, USA) with a P-value of