Image pro 4

Two cochleae were used that had been previously processed and morphologically analyzed (Rask-Andersen et al. 2000 (link); Table 1). Maximal resolution at this voltage was estimated to approx. 2 nm. Digital photos were taken at 1,280–1,024 ppi resolution. Measurements were performed using image analysis software Image Pro 4.5.1.29 (Media Cybernetics, MD, USA). BM widths were calculated from the tympanic lip to the basilar crest. The ratio between BM and scala tympani width assessed parallel was also derived. BM thickness was evaluated at three different locations: near the spiral ligament insertion (SL), under the outer pillar foot (OPF) and at spiral lamina insertion (OSL). The TCL thickness was estimated beneath inner and outer hair cells. Measurements were made at different cochlear turns. The decellularized human temporal bone was critical point dried with carbon dioxide, coated with gold-palladium in a sputter coater and imaged using a Hitachi S3500N Variable Pressure Scanning Electron Microscope at the University of Minneapolis.

Confocal laser scanning microscopy (CLSM) was used to explore the vitality of bacteria in the different depth layers of the biofilm. Bacteria were stained using a live/dead kit (Live/Dead BacLight viability kit, Molecular Probes, OR, USA) as described before [20] (link). Briefly, wells were washed, incubated for 15 min in a solution containing propidium iodide and SYTO 9 and washed again. To read the results directly, the wells were coated with emulsion oil to prevent dehydration. Fluorescence emission was detected using a Zeiss LSM 410 confocal laser scanning microscope (Carl Zeiss Microscopy, Jena, Germany). Red fluorescence was measured at 630 nm and green fluorescence at 520 nm; objective lenses: x60/oil, 1.4 numerical aperture. Horizontal plane (x-y axes) optical sections were made at 700 µm intervals from the surface outwards and images were displayed individually. The biofilm was quantified by measuring the area occupied by the bacteria with the aid of Image Pro 4.5 software (Media Cybernetics, Rockville, MD, USA).

5-bromo-2'-deoxyuridine (BrdU or BrdUrd) incorporation in MCF-7 and BT-474 cells was calculated by dual staining with human CK18 (rabbit polyclonal AP1021; Calbiochem, La Jolla CA) and BrdU (mouse monoclonal #347580; Becton-Dickinson, San Jose CA), followed by red Alexa-555 goat anti-rabbit and green Alexa-488 goat anti-mouse antibodies (Invitrogen). Basal MDA-MB-231 and BT-20 were stained for human CD44 (rabbit monoclonal 1998–1; Epitomics) or CK5 (rabbit monoclonal 2290–1; Epitomics) instead of CK18. For cells grown in conditioned media, BrdU quantitation was performed by immunocytochemistry (ICC) using Image J software. For 3D cultures immunohistochemistry (IHC) was used. Total cells were quantified by counterstaining with blue fluorescent 4’-6-diamidino-2-phenylindole (DAPI). Antibody against phosphorylated Histone H3 (Rabbit pAb Millipore # 06–570) was used for IHC as described [30 (link)].
Proliferation rates were calculated by the ratio of BrdU + nuclei (green) to DAPI + nuclei (blue) in CK18+, CD44+ or CK5+ cells (red) using Image Pro 4.5 software (Media Cybernetics). Quantification of BrdU incorporation and phosphorylated Histone H3 assays were performed in a minimum of five different fields from three independent experiments.