Fsx100 inverted microscope

Gram staining was conducted using a commercial kit (Nissui Co., Tokyo, Japan); the stained samples were observed using a FSX100 inverted microscope (Olympus, Tokyo, Japan). Cell viability in a co-aggregate was evaluated in a 3- to 5-h co-culture of L. casei and GFP-expressing E. coli. The cells were stained with 3.5 µM propidium iodide (PI)^{44 (link)}. Fluorescence microscopy was performed using a Leica TCS SP5 confocal laser microscopic system (Leica Microsystem Co., Wetzlar, Germany). The excitation wavelengths used were: 395 nm for GFP and 488 nm for PI. The emission was observed at 509 nm and 633 nm, respectively. Images were acquired and analyzed using Leica LAS AF software, version 2.6.0.

Immunocytochemistry and CLSM were performed as previously described [74 (link),75 (link)]. Cells were cultured on 12 mm round coverslips coated with poly-D-Lysine/Laminin (BD Bioscience, Franklin Lakes, NJ). Cells were fixed with 4% paraformaldehyde for 20 min, then permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells were incubated in a blocking buffer containing 1% bovine serum albumin (for MZF1 and SCAND2) or, alternatively, 3% normal goat serum (for SCAND1) in PBS for 30 min, incubated with primary antibodies at 4 °C overnight and then with secondary antibodies at RT for 1 h in the blocking buffer. Cells were washed thrice with PBS for 5 min between the steps. Cells were mounted within ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Fluorescence images were acquired using Axio Vision CLSM (Zeiss, Oberkochen, Germany) with an AxioCam MR3 (Zeiss) camera for SCAND1, and alternatively, FSX100 inverted microscope (Olympus, Tokyo, Japan) for MZF1 and SCAND2. We used antibodies against MZF1 (C10502, Rb pAb, Assay Biotechnology, Fremont, CA), SCAND1 (ab64828, Rb pAb, Abcam), SCAND2 (5F1, H00054581-M02, Ms mAb, Thermo Fisher Scientific), and anti-rabbit IgG conjugated with Alexa Fluor 488 (Thermo Fisher Scientific).