Alterations in Airway Inflammation and Lung Function During Corticosteroid Therapy for Atopic Asthma: Immunohistology
Cryostat sections (6 |j,m in thickness) of the endobronchial biopsy samples were cut at — 25°C and placed onto poly-L-lysine-coated microscope slides. The slides were air dried for 60 min, fixed in chloroform/acetone (1:1), wrapped in cling film, and stored at — 20°C until used. Each biopsy sample was sectioned within 1 month of freezing. At least 40 sections were cut and stored at — 20°C until use. Representative sections of all samples were stained with 0.1% toluidine blue to reveal morphology and tissue integrity. Immunohistochemistry was employed to identify subsets of inflammatory cells. The indirect immunoperoxidase technique was used to identify T cells, using a cocktail of monoclonal antibodies (MoAbs) [MoAbs CD2, CD3, CD7, and CD8], macrophage/monocytes (MoAb CD68), and eosinophils (MoAbs EG1 and EG2).
A blinded investigator (L.W.P.) quantified cell numbers so mycanadianpharmacy. The presence, distribution, and quantity of cells were assessed using a computerized image analysis system (Solitaire; Seescan; Cambridge, UK). Three areas of epithelium and subepithelial connective tissue to a depth of 10 to 12 cells were measured in each section. Total areas of 12 X 104 |j,m2 were quantified on duplicate sections for each cell type in each biopsy specimen. Areas of each high-power field were determined with the image analyzer by drawing frames on the computer screen around the area to be quantified. These frames were designed to avoid damaged regions and areas of muscle and cartilage. The numbers of positive cells (brown by immunoperoxidase staining) within framed areas were point counted and all results were then reduced to cells per unit area by dividing the number of cells in each field by the area of section in squared micrometers calculated by the computer. Only nucleated cells were counted.