Spectral spinning disc confocal microscope

Direct imaging of tissues was performed using a Nikon/Spectral Spinning Disc Confocal microscope (X-1 Yokogawa spinning disc with Borealis modification). Ki67 staining was performed on paraformaldehyde-fixed paraffin-embedded liver tissue with heat-induced epitope retrieval in 10 mM sodium citrate buffer pH 6.0, rabbit anti-Ki67 (Abcam ab16667)
and DAKO Envision anti-rabbit HRP detection reagents.

The effect of the cranberry extract on structure of the polymicrobial biofilms was evaluated by confocal microscopic imaging as described previously [Klein et al., 2011] . Briefly, 1 µM Alexa Fluor 647labeled dextran conjugate (647/668 nm; Thermo Fisher Scientific, Scoresby, Australia) was added to the culture medium to label the EPS component of the biofilm during the biofilm growth phase. Biofilm microbial components were labeled at the end of the growth period using 1 µM SYTO 9 (480/500 nm; Thermo Fisher Scientific). The images were obtained using a spectral spinning disc confocal microscope (Nikon, Tokyo, Japan) at 5 randomly selected positions for each biofilm. Three dimensional (3D) renderings of biofilms and the quantification of EPS/microbial biovolumes was assessed with IMARIS 8.0 (Bitplane, Concord, MA, USA).
The biovolume was defined as the volume of the biomass (µm 3 ) divided by substratum surface area (µm 2 ).
The effect of the treatment and control solutions on biofilm structural architecture was assessed qualitatively from the confocal 3D images.