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. 2008 May;172(5):1155–1170. doi: 10.2353/ajpath.2008.070791

Figure 1.

Figure 1

Examples of results of in situ hybridization, IHC, and lectin staining in various organs of H5N1 autopsies. A: Lung tissue showing severe damage, hyaline membrane formation, edema, fibrin exudation, and cellular infiltration (H&E staining). B: Double labeling with in situ hybridization (NP anti-sense probe) (purple-blue signals) and IHC with antibody to tubulin β (red signals, arrowheads) show positive in situ hybridization signals in the cytoplasm of both a tubulin-negative nonciliated cell (arrow) and a tubulin-positive (asterisk) ciliated cell in the trachea. C: Positive IHC staining (anti-NP antibody) in the nuclei and cytoplasm of some pneumocytes (arrows). D: Double labeling with in situ hybridization (NP sense probe) and IHC with antibodies to surfactant antibody A showing both dark blue nuclear in situ hybridization signals (arrows) and brownish-red cytoplasmic IHC signals (arrowheads) in a single cell in the lung. E: Positive in situ hybridization signals (NP sense probe) in the cytoplasm of some cells (arrows) in brain tissue taken from the parietal lobe. Double labeling with antibodies to neuron-specific enolase identifies these cells as neurons (not shown). F: Positive IHC signals (anti-NP antibody) in large mononuclear cells (arrows) with morphological features of macrophages within the core of a chorionic villus (arrows). IHC with antibody to CD68 on consecutive sections shows that these cells are most likely Hofbauer cells (fetal macrophages) (not shown). G: Positive IHC signals (anti-HA antibody) in the cytoplasm of some pneumocytes in fetal lung tissue. H: IHC with antibodies to macrophage inflammatory protein-1α shows a large number of positive cells in lung tissue. I: Staining with Maackia amurensis lectin II (specific for α-2,3-linked sialic acids) detects the presence of avian influenza virus receptors on pneumocytes. A, C, D, and F involve tissues taken from a 24-year-old pregnant female infected with H5N1 virus who died 9 days after disease onset. B, E, H, and I are taken from a 35-year-old male H5N1 patient who died 27 days after disease onset. G is lung tissue of the fetus carried by the 24-year-old pregnant female. B, D, and E: The in situ hybridization probes were labeled with digoxigenin and a NBT/BCIP substrate chromogen kit (Promega Corp., Madison, WI) was used to visualize the in situ hybridization signals, resulting in a purplish blue color. Anti-HA and anti-NP antibodies were purchased from Beijing Perfect Biotechnology Ltd. (Beijing, China) and VivoStat Inc (Portland, ME), respectively. B–D and F: The IHC reaction products were detected with 3-amino-9-ethylcarbazole (AEC) (Sigma, St. Louis, MO), which gives a brownish-red color. G–I: The IHC reaction products were colorized with diaminobenzidine (Zymed Laboratories, South San Francisco, CA), which gives a brown reaction color. C and F–I are counterstained with hematoxylin. B and E are lightly counterstained with methyl green. Scale bars: 25 μm (A, C, D, F, G); 10 μm (B); 12.5 μm (E, I); 20 μm (H).