Quantifying cellular expression of cytochrome P450 1A1 (CYP1A1) in the pulmonary alveolus: a comparison of indirect enzymatic immunohistochemistry (IH) and indirect immunofluorescence (IF).
Battelli-LA; Hubbs-AF; Ma-JY; Kashon-ML; Castranova-V
Toxicologist 2001 Mar; 60(1):403
Two common immunologic detection systems, IH and IF were compared for use with morphometric assessment of CYPIAI induction in the alveolar region of the lung. Rats were treated intraperitoneally with either a known inducer of CYPIA1; i.e. 50 mg/kg p-naphthoflavone(NF) in corn oil (n=4), or corn oil alone (n=4). Lungs were perfusion-fixed with neutral buffered formalin and the left lateral lung lobe was paraffin embedded within 24 hours of collection. For IH, CYPIAI was localized using a streptavidin-biotin, peroxidase detection system and 3-amino-9ethylcarbazole as the chromogen. For IF, CYPIAI was localized using an Alexa 594 conjugated goat anti-rabbit antibody and cytokeratin 8 (a marker for type II cells in the alveolus) was localized using Alexa 488 conjugated donkey anti-sheep antibody. For both techniques, the primary antibody was a rabbit polyclonal antibody which reacted with rat CYPIA1. Four representative alveolar areas were captured by digital phoromicroscopy and images were analyzed using computerized morphometric analysis. The area of lung occupied by CYPIAI positive cells was significantly increased in NF injected rats using either technique to visualize CYPIAI containing cells. Using digital capture to minimize fading of fluorochromes, IF was significantly more sensitive than IH (P < 0.05). By colocalization analysis, most of the area containing CYPIA1 after induction was not located in an area also expressing cytokeratin 8. Thus, IF was able to localize the site of CYPIAI expression in the alveolus and indicated that most of the alveolar CYPIAI induction was not in the type II cells. Cellular morphology was better demonstrated by IH than by IF. Both IH and IF were useful in detecting an increased area of the alveolus involved in CYPIAI expression in formalin-fixed lung after exposure to the CYPIAI inducer, NF
Immunology; Laboratory-animals; Antibiotics; Computer-models; Cell-morphology
The Toxicologist. Society of Toxicology 40th Annual Meeting, March 25-29, 2001, San Francisco, California