Abstract
Our protocol describes immunofluorescent staining, hematoxylin and eosin staining and Masson’s trichrome staining on lung sections.
Keywords: Immunofluorescent staining, Hematoxylin and eosin staining, Masson’s trichrome staining, Frozen tissue sections, Paraffin-embedded tissue sections, Antibodies
Background
The primary function of the lungs is gas exchange. The lungs are composed of various specialized cells and tissues, including the bronchi, the bronchioles and the pulmonary alveoli to facilitate gas exchange. To study lung development or lung diseases in mouse models, the lungs can be removed from mice and either frozen and embedded in optimal cutting temperature (OCT) compound or chemically preserved and embedded in paraffin. To preserve lung tissue architecture, we filled the lung with 1 ml of OCT compound for preparing frozen sections, or 10% buffered formalin for paraffin sections (Zhou et al., 2016). Lung sections are then sliced from frozen or paraffin-embedded lungs and mounted onto slides for preparation of staining. We used frozen tissue sections for antibody-based immunofluorescent staining and used paraffin-embedded sections for hematoxylin and eosin (H&E) staining or Masson’s trichrome staining. Frozen sections are quicker to prepare for immunofluorescent staining and most antibodies work well on frozen sections. Paraffin sections can also be used for immunofluorescent staining, but they require deparaffinization, rehydration and antigen retrieval. Some antibodies do not work well on paraffin sections even after antigen retrieval. However, paraffin samples can be stored at room temperate for very long periods and can be easily cut into very thin sections. Paraffin sections preserve better tissue morphology than frozen sections, so they are better for H&E or trichrome staining. Immunofluorescent staining is a type of immunohistochemistry that makes use of fluorophores to visualize the location of the antibodies that specifically bind to their target proteins. H&E staining is the most widely used stain in histology and medical diagnosis. This staining method involves application of hemalum and eosin Y that color cell nuclei blue and cytoplasm pink to red. Masson’s trichrome staining is a three-color staining that is used for detecting collagen fibers in tissues. The staining produces blue collagen, dark brown to black cell nuclei and red background.
Materials and Reagents
Equipment
Procedure
Data analysis
Lung sectioning and staining are essential methods for studying lung development or lung pathology. Immunofluorescent staining visualizes the expression and localization of interested proteins. H&E staining is most widely used in histology studies and medical diagnosis. Masson’s trichrome staining detects collagen deposition in tissues. Figure 2 shows representative images of each staining on the lung sections from mice that had been undergone bone marrow transplantation and then infected with murine gamma herpesvirus 68 for three weeks. Figure 2. Lung section staining. The lungs were removed from C57BL/6 mice that had undergone transplantation of syngeneic EGFP-expressing bone marrow for 5 weeks, and followed by infection with murine gamma herpesvirus 68 (5 x 104 pfu/mouse) for 3 weeks. The lung had developed pneumonitis and pulmonary fibrosis as evidenced by infiltration of immune cells and deposition of collagen. A. Lung section is immunofluorescent stained with rabbit anti α-smooth muscle actin (α-SMA) antibody and α-SMA positive cells are visualized by Texas Red conjugated goat anti-rabbit IgG antibody in red. Donor bone marrow-derived cells express EGFP in green. DAPI counterstains all of the cell nuclei in blue. B. H&E staining colors cytoplasm pink and nuclei blue; C. Masson’s trichrome staining colors collagen blue, cytoplasm pink and nuclei dark brown to black.
Recipes
Acknowledgments
This protocol was adapted from our publication (Zhou et al., 2016). This work was supported by NIH grants AI117229, HL115618, T32HL07749 and 2UL1TR000433.
References
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