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

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.

Persistent G. lamblia impairs growth in a murine malnutrition model

“…At the time of sacrifice, 1.5-to 2-cm segments of small bowel (duodenum [4 cm after the pylorus], jejunum [8 cm distal to the duodenal segment], and ileum [1 cm proximal to the cecum]) were fixed in 10% Zn-formalin, transferred to 70% EtOH prior to paraffin embedding, and then stained with H&E. Villus length and crypt depth measurements were recorded only on full-length villi with adjacent crypts, defined as a "villus-crypt unit" (7-15 villi per mouse) (Image J software; NIH) and read in a blinded manner (L.A. Bartelt and J. Roche). Eosinophil morphology was confirmed on a subset of tissues using Congo red stain (62). Rabbit anticleaved caspase-3 antibody staining was performed using a DAKO auotostainer with a hematoxylin counterstain according to protocol.…”

Respiratory syncytial virus-like nanoparticle vaccination induces long-term protection without pulmonary disease by modulating cytokines and T-cells partially through alveolar macrophages

“…The scores were in a range of 0 (no inflammation) to 3 (severe inflammation) following the criteria of the scoring system. 24 PAS-positive areas were calculated using the Photoshop program.…”

Tumor-Associated Tissue Eosinophilia in Squamous Cell Carcinoma of Head and Neck

“…It is defined that Sirius Red staining has higher specificity and contrast to show eosinophils compared to Luna, Congo red and hematoxylin and eosin staining (12). Another reason for this difference of eosinophil count with other studies could be the counting method.…”

“…Hematoxylin and eosin stained slides were reevaluated and their diagnosis was confirmed by an oral and maxillofacial pathologist. Sections of paraffin-embedded blocks were stained with histochemical staining of Sirius red (12). With an optical microscope (Nikon, Japan), all eosinophils were counted in 10 high power fields with magnification of 400x (eosinophil/10HPF).…”