Anti biotin fitc

S. montanus was grown at 27 °C for 18 h in an Armbruster medium [23] with agitation. The culture (3 mL) was aseptically mixed with 50 μL of 10 mM NHS-LC-LC-Biotin (Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 30 min at 25 °C. The N-biotinylated filaments of S. montanus were trapped using an Ultrafree-CL filter unit (5 μm, Merck), washed with 25 mM HEPES-NaOH buffer (pH 7.2) containing 1 mg/mL of bovine serum albumin, and then suspended in 2 mL of the same buffer. The suspension (0.5 mL) was dropped onto an autoclaved glass slide (Frontier coat, Matsunami, Osaka, Japan) and allowed to stand at 25 °C for 20 min. The glass slide with Nbiotinylated filaments deposited on it was washed with the buffer and subsequently immunostained with a fluorescein isothiocyanate (FITC)-conjugated anti-biotin antibody (anti-Biotin-FITC, Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min. After washing with the buffer, phase-contrast and epifluorescent microscopic observations were performed using a BX51 microscope equipped with a U-MNIBA3 mirror unit (Olympus, Tokyo, Japan). For selective visualization of pre-existing regions of the sheath, N-biotinylated filaments fixed on the glass slide were allowed to grow in Armbruster medium at 25 °C for 3 h. After cultivation, the filaments were immunostained for microscopy.