Confocal software lite

The presence of Ki67-positive tumor cells and CD31-positive vessels in rat stroma tissue was analyzed using a fluorescence laser scanning system (TCS-SP2 scanning system and DM IRB inverted microscope, Leica®, Solms, Germany). Ki67 and CD31 staining were performed with specific antibodies as described above. Dye-coupled Alexa antibodies (Alexa-488 for Ki67 in green and Alexa-647 for CD31 in red; Molecular Probes, Eugene, USA) were used as secondary antibodies. Subsequently, Hoechst 33342 was used for labeling of nuclear DNA (Sigma®, Taufkirchen, Germany). Leica Confocal Software Lite (Leica®, Solms, Germany) was used for evaluation according to the manufacturer’s instructions.

Synovial tissues with ELSs from RA patients and tonsils from patients undergoing tonsillectomy were embedded in O.C.T (Sakura) and 10 μm cryo-sections were cut and fixed in ice cold acetone. In addition, cytospin preparation of RA synovial fibroblasts and NG2⁺ pericytes were prepared. The slides were rehydrated and blocked with 2% horse serum (Jackson ImmunoResearch, 008-000-121) in a humidified chamber, incubated at room temperature with primary Abs for 2 h, followed by 3X wash in PBS then incubation with secondary Abs for 1 h. After incubation with the secondary Abs, the slides were washed in PBS 3X, dried and mounted with Vectashield Antifade Mounting Medium (Vector Laboratories), cover-slipped, and examined with a Leica TCS SP2 AOBS confocal laser-scanning microscope. Four lasers (405, 488, 543, and 633 nm) were used and far-red emission was converted into pseudo-color. Parameters were adjusted to scan at 1,024 × 1,024-pixel density and 8-bit pixel depth. Emissions were recorded in separate channels, and digital images were captured and processed with Leica Confocal Software Lite. The Abs used in this study are listed in Table 2 and their concentrations ranged between 5 and 10 μg/ml.

To analyze the biofilm structure and thickness, 24-h biofilm-embedded cells in PBS were stained with Syto-9 dye from the BacLight bacterial viability kit (Invitrogen, Cergy Pontoise, France) and observed with Confocal Leica DMR TCS SP2 (Leica microsystems, Wetzlar, Germany, with magnification of 10×) fitted with water immersion dipping lenses. The excitation wavelength at 488 nm (blue laser) generated green fluorescence and all light rays emitted above 500 nm were collected. Biofilm structure was analyzed by taking a series of horizontal sections (stacks) to evaluate its thickness. All images were processed using Leica Confocal Software Lite.