Immunohistochemistry
Antibodies for detection of inflammatory cells, including neutrophils and mast cells, were purchased from Dako (Glostrup, Denmark). Human anti CD3, which is a T cell marker, and human anti ECP (EG2), an eosinophil marker, were purchased from Serotec (Oxford, UK) and Diagnostic development (Uppsala, Sweden), respectively. Antibodies used to detect transcription factors and detoxification enzymes, including p–c-Jun, NQO1, c-Fos (mouse monoclonal antibodies), Nrf2 and AHR (rabbit polyclonal antibodies), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The supplier for the antibodies against Cyp1A1, Cyp1B1, EPHX (mouse monoclonal antibodies) and Aldo–keto reductases (AKR1A1, AKR1C1 and AKR1C3) was Abcam (Cambridge, UK). Biotinylated rabbit anti-mouse and swine anti-rabbit antibodies were purchased from Dako.The staining procedure has been previously described in detail [Quantification of the immunohistochemical staining and other analyses were performed by one person who was unaware of how the coded samples corresponding to the exposures. All analyses were done double blinded, with codes broken only after completion of the full analysis and statistics. When quantifying the immunohistochemical staining, a light microscope was used to count inflammatory cells according to their immunoreactivity with specific antibodies in the epithelium and the submucosa respectively, excluding mucosal glands, blood vessels and smooth muscle. A computer-assisted image analysis program (Leica Q500IW, Leica Cambridge UK) was used to calculate the length of the epithelium and the area of the submucosa. Cell counts were expressed as cells/mmImmunoreactivity within the bronchial epithelium. Upper left shows immunoreactivity to NQO1 post-air exposure and upper right shows NQO1 post-DE exposure. Middle panel shows immunoreactivity to AhR antibody, middle left, post-air exposure and middle right, post-DE exposure. Immunoreactivity to P–C-Jun antibody is shown in lower panel, with lower left at post-air exposure and lower right at post-DE exposure. Narrow arrows indicate positive intracytoplasmic staining and bold arrows indicate nuclear staining. Scale bars = 50 μm (micro meters)Due to lack of double staining or sequential sections for co-localization, we were not able to quantify nuclear translocated AhR in specific cells in the submucosa. Therefore, AhR activation in the submucosal leukocytes was expressed as the number of nuclear translocated AhR in the leukocytes/mm