Assessment of Lung Eosinophils In Situ Using Immunohistological Staining
Eosinophils are rare white blood cells that are recruited from circulation to accumulate in the lung in mouse models of allergic respiratory inflammation. In hematoxylin–eosin (HE) stained lungs, eosinophils may be difficult to detect despite their bright
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Introduction Eosinophils are considered the hallmark cell that mediates destructive [1–3] and immune regulating [4–8] activities in asthma pathologies [9–11]. To analyze eosinophils in situ, lung sections are often used as a measure of their numbers and states of activation in allergic respiratory pathology. Allergen models of pulmonary inflammation induce many characteristics of human pathology including increased mucus secretion, smooth muscle thickening, airway inflammation, and eosinophilic infiltration [12, 13]. Although evidence of degranulation is controversial in most acute allergen models [14, 15], some chronic models develop significant release of granule proteins in the lungs [16], a feature found in human asthmatic lung biopsies and in lung injury [17]. These pathologic changes are often viewed with use of
Kumi Nagamoto-Combs (ed.), Animal Models of Allergic Disease: Methods and Protocols, Methods in Molecular Biology, vol. 2223, https://doi.org/10.1007/978-1-0716-1001-5_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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standard dyes that characterize inflammation (HE), mucus production and goblet cell metaplasia, periodic acid–Schiff (PAS) or collagen deposition as with Masson’s trichrome or picrosirius red. In order to identify eosinophils in situ, the lungs of euthanized mice are often formalin-fixed, embedded in paraffin, and thinly sliced (5 μm) onto glass slides. These slides are then deparaffinized and stained with dyes meant to highlight eosinophils based on the unique nature of their granule proteins. The most common dyes are acidic and chosen due to their tendency to stain cationic eosinophil granule proteins. In short, eosinophils are eosin-philic (i.e., eosinloving), due to the acidic eosin dye accumulating on the highly positively charged and acidophilic granule proteins as discovered by Dr. Paul Ehrlich over a century ago [18]. Additional dyes that are used for identifying eosinophils include Congo red and Luna. With these dyes, however, distinction between neutrophils and eosinophils is challenging, and nonspecific staining is present. For these reasons, they tend to produce less specific staining than immunohistochemistry (IHC) or immunocytochemistry (ICC), which in contrast utilizes antibodies that recognize eosinophil-specific antigens [19]. Although eosinophils can be identified by dyes, they must be differentiated manually with a trained eye. These dyes are commercially available, have easily accessible instructions on their use, and will not be discussed in this chapter. Immunostaining assays such as IHC, ICC, and immunofluorescence (IF) use antibodies that recognize specific proteins of interest [20]. Monoclonal antibodies are superior to polyclonal antibodies due to their specificity for unique epitopes (for reviews [21–23]). Secondary granule proteins in mouse eosinophils include eosinophil peroxidase (EPX), major basic protein (MBP-1), and the divergent homologs mouse ribonucleases (mEARs) that are related to h
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