Figure 3. Localization of Td_neu.

(A) Immunoblotting analysis of proteinase K-treated Td₃₅₄₀₅ whole-cell lysates. The spirochetes were either incubated with proteinase K (240 μg/ml) or PBS at 37°C for 1 h. The resultant samples were separated on SDS-PAGE and then probed with αTd_neu and αFlaA. (B) IFA analysis. Td₃₅₄₀₅ and Tde471^mut cells were fixed with methanol, stained with αTd_neu, and counterstained with a goat-anti-rat Texas red antibody as previously described. The micrographs were taken under DIC light microcopy or fluorescence microscopy with a tetramethylrhodamine isothiocyanate (TRITC) emission filter, and the resultant images were merged. (C) Detection of Td_neu in the supernatants prepared from the cultures of Td₃₅₄₀₅, Tde471^mut, and Tde471^com strains by immunoblotting analysis. FlaA was used as a sample preparation control. (D) Detecting neuraminidase activity in the supernatants by the filter paper spot test as described in Figure 1A.