Quantifying Airway Mast Cells in Lung Tissue

Fixed lung samples were stained for mast cells which were quantified and visually differentiated to the medium sized airways (0.5–5.0 mm) and parenchyma, and normalized to total tissue area. Before beginning any staining, slides with 7 μm sections were deparaffinized, rehydrated, and washed in distilled water. Slides were stained with hematoxylin and eosin in order to visualize lung structure. Mast cells were detected using both the glycoprotein avidin (conjugated to HRP, Vector Laboratories, Burlingame, CA, United States) (28 (link), 29 (link)) and toluidine blue, which is the classic metachromatic stain for mast cells (30 (link), 31 ). Conjugated avidin is a well-defined histochemical method for identifying human mast cells, which express heparin proteoglycans in the secretory granules (32 (link)), binding specifically to the proteoglycan granules (28 (link), 29 (link)). Deparaffinized sections were exposed to avidin-HRP (1:500 dilution) and then NovaRED (Vector Laboratories, Burlingame, CA, United States) was used as the chromogen substrate. Each lung section was analyzed for mast cells and quantified as cells per square millimeter of lung tissue. For all histological analyses, de-identified tissue sections were viewed under transmitted light with either a 10X or 60X objective and an inverted epifluorescence microscope (Nikon Eclipse TE 2000-U, Melville, NY, United States) interfaced to a SPOT Insight 2 megapixel color camera (Diagnostic Instruments, Sterling Heights, MI, United States). Whole slide image specimens were scanned and stored on the imaging work station as routinely performed in the lab. When quantifying cell number and determining areas of tissue, Metamorph software (version 6.2; Universal Imaging, West Chester, PA, United States) was used. All histological analyses and cell number quantifications were performed by two investigators in a blinded fashion.