Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 11/6/2000
Publication Date: N/A
Technical Abstract: Anionic peptide is a homopolyaspartate peptide with antimicrobial activity. It was first identified in lung of sheep and recently detected in respiratory cells of man and cattle. Recent work in our laboratory has demonstrated that immature (hyperplastic) respiratory cells in chronic pneumonia of lambs have increased intensity of anionic peptide immunoreactivity by immunohistochemistry. In order to quantify the level of anionic peptide in these cells, we adapted a procedure to retrieve the hyperplastic cells from ovine lung tissue using a laser capture microdissection system (Pixcell II, Arcturus, Inc.). Bronchiolar and alveolar epithelial cells were captured from both pneumonic and normal lungs that were either: 1) fixed in formalin, processed routinely, embedded in paraffin and sectioned; or 2) frozen in OCT embedding compound. In the capture process, surrounding tissue (lung cartilage, vessels, and connective tissue) remained on the section and was, thereby, separated fro the retrieved epithelial cells. For optimal retrieval of epithelial cells, formalin-fixed and frozen sections required plain (non-coated) glass slides, fresh xylene (non-recycled), and the absence of glues or other adhesives (e.g., gelatin, bovine serum albumin) in microtome waterbaths. Tissues sectioned onto slides required storage in a dehydrated environment (dessicator). Proteins from captured cells were released by incubation of the cells with a lysis buffer and sonication. This work demonstrates that individual respiratory epithelial cells can be removed from ovine lung sections without the surrounding lung tissue by laser capture micro- dissection. Proteins, such as anionic peptide, can be assessed from these captured cells by Western Blots, ELISA, Dot Blot, or other assays.