Concanavalin a alexa fluor 647 conjugate

For bright-field microscopy analysis, 5 μl of cell suspension at the indicated time points were examined under a NIKON TI-E inverted fluorescence microscope (Nikon, Japan) in differential interference contrast (DIC) mode. Immunofluorescence microscopy analysis was carried out as previously described (28 (link)). Briefly, S. islandicus REY15A cells were collected and pelleted down at 5000 g for 5 min, re-suspended in 300 μl PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄·12H₂O, 2 mM KH₂PO₄, pH 7.4), and fixed by addition of 700 μl cold absolute ethanol and kept at 4°C for at least 2 h. The fixed cells were washed for 3 times with PBST (PBS plus 0.05% Tween-20) to remove ethanol. Primary antibodies against ESCRT-III (HuaAn Biotechnology Co., Hangzhou, Zhejiang, China) were added with a dilution of 1:1000 in PBST and incubated at 4°C overnight. The cells were washed 3 times and then incubated with the goat anti-rabbit secondary antibodies Alexa Fluor® 488 (1:1000, Thermo Fisher Scientific, USA) for ESCRT-III, and Concanavalin A Alexa Fluor 647 Conjugate (50 μg/ml, Invitrogen™, Thermo Fisher Scientific, USA) for S-layer, and kept at 4°C for 2–4 h. The localization of ESCRT-III was observed under a SP8 confocal microscope, and the data were analysed using Leica Application Suite X (LAS X) software (Leica).