Basal-Like Cells Constitute the Proliferating Cell Population in Cystic Fibrosis Airways

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1 Basal-Like Cells Constitute the Proliferating Cell Population in Cystic Fibrosis Airways Judith A. Voynow, Bernard M. Fischer, Bruce C. Roberts, and Alan D. Proia On-line Data Supplement

2 1 Methods Image Analysis. Digital images from randomly selected, ten separate fields of airway epithelia per subject were captured using an Optronics DEI 750 D digital output unit (Optronics, Goleta, CA) mounted on an Olympus BX40 microscope (Opelco, Dulles, VA) and analyzed by the image analysis software package Optimas (Media Cybernetics, Silver Spring, MD). Images were captured using a 60x objective lens. A calibration image was captured at the same magnification from a micrometer slide (Graticules Ltd., Tonbridge, Kent, England). For calculation of the proliferation index, the total number of nuclei within the captured image was counted manually, as was the number of nuclei staining with MIB-1 antibodies. We calculated the ratio of positively staining nuclei to total nuclei in the ten fields, and the average was used as the proliferation index for that patient. Immunohistochemistry. Methods for antigen retrieval and primary antibody detection varied slightly for each primary antibody. Negative controls employed non-immune IgG (for example mouse IgG from Southern Biotechnology Associates, Birmingham, AL for Ki-67, cytokeratins 5/14, and EGFR immunohistochemistry). Immunohistochemistry was performed for Ki67, cytokeratins 5/14, EGFR and ErbB2. Ki-67. Immunoperoxidase staining of proliferating cells used MIB-1 monoclonal antibody (DakoCytomation, Carpenteria, CA), which recognizes the Ki-67 antigen in cycling cells. Ki-67 nuclear antigen is expressed throughout the cell cycle (G 1, S, G 2 and M phases) but not in resting cells (G 0 ). Immunostaining was performed after antigen retrieval by heating the unstained sections in 10 mm citrate buffer, ph 6.0, at 95 C for 20 min. After antigen retrieval, the slides were rinsed three times with phosphate-buffered

3 2 saline (PBS) and then incubated for 15 minutes at room temperature in 5% normal goat serum. The primary antibody was diluted 1:25 in Primary Antibody Diluting Buffer (Biomeda Corp., Foster City, CA), with incubation for one hour at room temperature. Following washing three times in PBS, the MIB-1 labeling was detected using a Super Sensitive Link-Label IHC Detection System with diaminobenzidine (DAB) chromogen (BioGenex Laboratories, San Ramon, CA). Slides were counterstained with 1.5% methyl green in 0.1M acetate buffer, ph 4.6 (Sigma Aldrich, St. Louis) and coverslipped. In ten randomly selected fields of airway epithelia, total nuclei and total MIB-1-positive nuclei were counted to determine the percentage of MIB-1 positively-stained nuclei. The percentage of MIB-1 positively-stained nuclei is a reflection of the proliferating cell population. Control groups with young subjects (mean age 9.3 years, n=6) or with older subjects (mean age 67 years, n=9) had identical indices of proliferation (unpublished observation, A.D.P.) supporting the concept that differences in age do not affect the proliferative activity of normal airway epithelia. Cytokeratins 5/14. Antigen retrieval was accomplished by incubation with 0. 25% pepsin (Sigma Chemical, St. Louis, MO) in 0.1 N HCl, 14 min, 40 C. After rinsing twice with PBS, slides were incubated with primary antibody, mouse anti-human high molecular weight cytokeratins (clone 34βE12, DakoCytomation) at 1:25 dilution, 60 min, room temperature. 34βE12 antibodies react with the high molecular weight antigen pair 1/10 and the intermediate molecular weight pair 5/14; the 1/10 pair is present only in stratified squamous epithelium, while the 5/14 pair is useful for identifying basal and parabasal cells of the human airway epithelium (E1). Antibody detection used a

4 3 Histostain -Plus Kit with DAB chromogen (Zymed Laboratories, South San Francisco, CA). The slides were counterstained with hematoxylin. Epidermal growth factor receptor (EGFR). Epitope retrieval was performed by digesting the section with Protease XXV lyophilized powder, reconstituted to 2 ml immediately before use; NeoMarkers Lab Vision Corporation, Fremont, CA), 5 min, 37 C. The primary antibody, Ab-10, a mouse monoclonal anti-human EGFR IgG1 that recognizes the 170 kd wildtype epitope (clone 111.6, NeoMarkers) was used at concentration 4 µg/ml with an incubation of 1 h, room temperature. Detection of antibody binding was performed using an avidin-biotin complex method (VECTASTAIN Elite ABC kit, Vector Laboratories, Burlingame, CA). ErbB2. Epitope retrieval was performed by incubating slides in 10 mm citrate buffer, ph 6.0, 10 min, 95 C. The primary antibody, rabbit anti-human c-erbb2 polyclonal antibody (A0485, DAKO) was used at a dilution of 1:100 with an incubation of 1 h, room temperature. The Histostain -Plus Kit with DAB chromogen (Zymed Laboratories) was used as the detection system.

5 4 References E1. Boers, J., Ambergen, AW, Thunnissen, FBJM Number and proliferation of basal and parabasal cells in normal human airway epithelium. Am. J. Respir. Crit. Care Med 157: