Eclipse ti u confocal microscope

The SNS segments were stained with 3.3 µM SYTO 9 (Invitrogen, Life Technologies, Eugene, OR, USA) and 10 µM propidium iodide (PI) (Sigma) in PBS for 30 min [26 (link)] and visualized by a spinning disk confocal microscope (Nikon Yokogawa W1 Spinning Disk, Tokyo, Japan, with 50 µm pinholes) or a Nikon eclipse Ti-U confocal microscope (Nikon Instruments, Tokyo, Japan). The biofilm depth was assessed on spinning disk microscope by capturing optical cross-sections at 2.5 μm intervals from the bottom of the biofilm to its top. The SYTO 9 green fluorescence dye, which enters both live and dead bacteria, was visualized using 488 nm excitation and 515 nm emission filters. The PI red fluorescence dye, which only penetrates dead bacteria, was measured using 543 nm excitation and 570 nm emission filters. Thus, live bacteria fluoresce green light, while dead bacteria fluoresce both green and red light. Three-dimensional images of the formed biofilms were reconstructed using the NIS-Element AR software. This software was also used to analyze the fluorescence intensity of SYTO 9 and PI staining in each captured layer of the biofilms. The percentage of total biomass of viable cells in biofilm formed in the presence of SRV-CHX was calculated in comparison to SRV-placebo.

Cells were incubated on plates coated with 3 µg/cm² poly-D-lysine (EMD Millipore, Billerica, MA), fixed in 10% formalin (Sigma). For Nile red staining, fixed cells were washed three times in 1× PBS, and treated for 10 minutes at 37 °C with a 1 µM Nile red solution prepared in DMSO. Cells were then washed once with 1× PBS, counterstained with DAPI (4′,6-diamidino-2-phenylindole) and washed again with water. For Oil Red O staining, fixed cells were treated with a filtered 0.5% Oil Red O (Sigma) solution prepared in isopropanol. Cells were then washed with deionized water and pictures were taken using Nikon Eclipse Ti-U confocal microscope (Nikon, Melville, NY) fitted with a QiCam (Qimaging, Surrey, BC). LDs were quantified by manual counting using ImageJ software^{40 (link)}. For Oil Red O quantitation, stained cells were dried and Oil Red O was eluted in isopropanol with gentle shaking. Eluted Oil Red O was quantified in supernatants by spectrophotometry at 500 nM.

Immunofluorescence staining was performed as described previously [87 (link)] using anti-Rab7 (diluted 1:100, Cell Signaling Technology, Danvers, Massachusetts, USA), anti-TLR7 (diluted 1:500, Novus Biologicals, Littleton, Colorado, USA), and Alexa Fluor 488 goat anti-rabbit IgG (diluted 1:2000, Thermo Fisher Scientific) as primary and secondary antibodies, respectively. After DNA counterstaining with ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific), images were acquired using a Nikon Eclipse Ti-U confocal microscope (Melville, New York, USA) at the Bioimaging Facility of the Sidney Kimmel Cancer Center at TJU.