A novel subset of putative stem/progenitor CD34(+)Oct-4(+) cells is the major target for SARS coronavirus in human lung

Chen, Yongxiong; Chan, Vera Sau-Fong; Zheng, Bojian; Chan, Kelvin Yuen-Kwong; Xu, Xiaoning; To, Leo Yuk-Fai; Huang, Fang-Ping; Khoo, Ui-Soon; Lin, Chen-Lung Steve

Sequential triple immunostaining on paraffi n sections. Sequential immunostaining was performed on 5-m paraffi n sections of formalin-fi xed lung samples. Samples were de-paraffi nized and rehydrated. After blocking endogenous peroxidase with 0.3% H 2 O 2 , 0.03% NaN 3 for 30 min at room temperature, mouse anti -surfactant A antibody was added at 4 ° C overnight. Sections were then incubated with peroxidase-conjugated EnVision plus reagent (anti -mouse, ready to use; DakoCytomation) for 1 h at room temperature and developed with diaminobenzidine chromogen substrate (DakoCytomation). Sections were subjected to microwave heating to block endogenous alkaline phosphatase as well as to inactivate the antibody from the fi rst staining. Mouse anti-SARS CoV nucleocapsid antibody was applied at 4 ° C overnight, followed by anti -mouse universal immuno-alkalinephosphatase polymer (ready to use; Nichirei Corporation) for 2 h at room temperature. The color was subsequently developed with a fast red substrate system (Sigma-Aldrich). After immunohistochemical staining, sections were again microwaved to inactivate the antibody from the second staining, followed by incubation with mouse anti-cytokeratin AE1/AE3 at 4 ° C overnight. Sections were incubated with FITC-conjugated goat anti -mouse IgG (1:400; Sigma-Aldrich) at room temperature for 2 h. Sections were mounted with medium for fl uorescence with DAPI (Vector Laboratories). Electronic images of the immunohistochemical and immunofl uorescence staining, visualized under a fl uorescence microscope (eclipse E600; Nikon), were captured and saved to a computer using the software ACT-1 (Nikon). IHC, FISH, and ISH on paraffi n sections. Sequential IHC was performed similarly as described above. After immunostaining, sections were treated with 0.2 N HCl for 30 min at room temperature to block alkaline phosphatase activity from the previous staining. Sections were then digested with 10 g/ml proteinase K at 37 ° C for 15 min. Subsequent FISH and ISH were performed on the same section as described previously ( 13 ) . The same fi eld as in the IHC, FISH, and ISH images were selected and visualized under the same microscope and saved as electronic images for comparison and analysis of colocalization.