A new approach to clearly identify collagen fibers/fibrils and inflammatory cells from H&E stained murine lung sections using fluorescent microscopy

Hematoxylin‐eosin (H&E) stains are commonly used in lung histology. Tissue stained with H&E was found to be revealed fluorescence. However, it is paucity in study of the lung using this advantage of fluorescent imaging technique. In this study, we used female mice (C57BL/6, 9–11 weeks) as the model for intratracheal administration of bleomycin (3.33 U/kg) or saline. The lung was harvested and prepared for paraffin embedded or cryostat frozen sections. We imaged the H&E stained lung sections using Zeiss fluorescent microscope (Axioplan 2) with a color digital camera (AxioCam MRc). We found that fluorescent images of H&E‐stained lung sections provide more precise anatomic structure of tissue such as collagen fiber/fibril accumulation associated with bleomycin‐induced leukocyte extravasation in pulmonary muscular vessels, which was matched with collagen fibers stained using Masson's trichrome stain. We could identify muscular layers, basement, and endothelium or epithelium in the vascular or bronchial walls in the lung with different fluorescence in H&E stained lung sections, which is less clear visualized in the bright‐field images. Also, the cells such as red blood cells, macrophages, and inflammatory cells including neutrophils, eosinophils, and lymphocytes can be easier to be identified in the lung sections based on their morphologic features of the nucleus and cytoplasm with a dark background using fluorescent microscope. The results suggested that fluorescent images combined with bright field images of H&E and Masson's Trichrome stained lung sections may provide more detailed information for collagen deposition and cell identification in the lung.