Comparison of Histochemical Methods for Murine Eosinophil Detection in an RSV Vaccine-enhanced Inflammation Model

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Abstract
A comparative study of histochemical detection of eosinophils in fixed murine tissue is lacking. Five histochemical methods previously reported for eosinophil detection were quantitatively and qualitatively compared in an established murine RSV vaccine–enhanced inflammation model. Nonspecific neutrophil staining was evaluated in tissue sections of neutrophilic soft tissue lesions and bone marrow from respective animals. Eosinophils had granular red to orange-red cytoplasmic staining, depending on the method, whereas neutrophils had, when stained, a more homogenous cytoplasmic pattern. Nonspecific background staining of similar coloration was variably seen in vascular walls and erythrocytes. Astra Blue/Vital New Red, Congo Red, Luna, Modified Hematoxylin and Eosin, and Sirius Red techniques were all effective in detecting increased eosinophil recruitment compared to controls; however, differences in eosinophil quantification varied significantly between techniques. Astra Blue/Vital New Red had the best specificity for differentiating eosinophils and neutrophils but had a reduced ability to enumerate eosinophils and was the most time intensive. The Luna stain had excessive nonspecific staining of tissues and a reduced enumeration of infiltrating eosinophils, which made it suboptimal. For multiple parameters such as eosinophil detection, specificity, and contrast with background tissues, the Sirius Red followed by Congo Red and Modified Hematoxylin and Eosin methods were useful, each with their own staining qualities.

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