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. 2005 Sep 15;33(6):589–600. doi: 10.1165/rcmb.2005-0213OC

Figure 6.

Figure 6.

Characterization of human bronchus epithelium in primary cultures. (A) (Left) Representative histopathology of human bronchial biopsy stained with H&E. All biopsies were fixed in 10% buffered formalin and embedded in paraffin wax. Paraffin blocks were sectioned according to a standard protocol at 5 μm, and serial sections were mounted in consecutive order on silane-coated glass slides (Superfrost-plus; Fisher Scientific, Pittsburgh, PA). A total of 12 slides containing 8 sections/slide were prepared from each paraffin block. Slides 4 and 9 were stained with H&E, and epithelial abnormalities were classified according to recent World Health Orgaization criteria as normal, reserve cell hyperplasia, squamous metaplasia, or dysplasia (low or high grade). The presence or absence of ASD, as defined subsequently, was noted in each biopsy. Two examiners evaluated the specimens, and joint review and consultation resolved diagnostic disagreements. (Right) Representative cloning in soft agar of one bronchoalveolar cell primary culture. H358p53+/+ cells were the positive control. (B) Representative immunocytochemical staining of primary cultures. Mouse antihuman cytokeratin monoclonal antibodies (mAbs) RCK102 (1:25; Organon Teknika, Boxtel, Holland), NCL5D3 (1:100; Organon Teknika), and 34βE12 (1:100; Dako Corp., Carpinteria, CA) were used to confirm epithelial phenotype. Immunostaining for vimentin using mouse antihuman vimentin mAbs (1:100; Organon Teknika) was performed to exclude contamination by fibroblasts; antihuman cluster of differentiation (CD) 3 mAbs (1:200; Dako, Glostrup, Denmark) to exclude lymphocyte contamination; and antihuman CD68 mAbs (1:100; Dako) to exclude macrophage contamination in cultures. (C) Effect on p53 protein levels. Cells in primary cultures were treated for 2 h with CPT 10 μM, then proteins were extracted, and p53 protein level increase was evaluated by Western blot. Lanes 1–6: control (untreated cells); 1a–6a: cells treated with CPT. Data are representative of three replicate experiments yielding similar results.