The tubing was carefully peeled away from the frozen biofilm by warming up the tube part briefly between fingers. The frozen biofilm sample was dipped vertically into the center of a cryosectioning cup filled with fresh OCT which was placed RGFP966 nmr on dry ice until it was completely frozen. Frozen samples were sectioned at -19°C using a Leica CM1850 cryostat. The 5 μm thick cryosections were mounted on Superfrost/Plus microscope slides (Fisher Scientific), washed gently with distilled water to remove the excess OCT and dried at room temperature. Cryosections were imaged using a Nikon Eclipse E800 microscope interfaced to a Metaview 2.0 image acquisition system (Molecular

Devices). Unstained sections were viewed in transmission using DIC optics. Sections stained with calcofluor (Fungi-Fluor™ stain, Polysciences, Inc) were viewed in epi-fluorescence mode. Antibody labeling of (1,3) β glucan in biofilm cryosections The selleckchem protocol for APR-246 mouse staining biofilm cryosections for (1,3) β glucan was a modification of a published protocol [77]. The primary monoclonal antibody (mAb) was from Biosupplies Australia (produced in mice). The secondary anti-mouse antibody, conjugated to Alexa

Fluor 488, was from Invitrogen (produced in rabbits). We used planktonic cells grown at 30°C and adhered to slides used for cryosectioning (Superfrost/Plus microscope slides, Fisher Scientific) as positive and negative controls. The negative control was omission of the primary antibody. In this case no fluorescence was detected under exposure conditions EGFR inhibitor in which there was relatively bright fluorescence originating from cells exposed to the primary antibody. In addition, fluorescence was in every case associated with cells as confirmed by comparing images acquired using epi-fluorescence and transmission modes (data not shown). OCT was rinsed from the biofilm cryosections

before antibody staining using Tween Tris Buffered Saline (TTBS), pH 7.6. This was followed by exposure to TTBS with 1% BSA (15 min), exposure to the primary mAb at 4 ug per ml in TTBS (1 h), three washes with TTBS (5 min each), exposure to the secondary Ab at a 1:100 dilution in TTBS (30 min) and a wash with TTBS 3 times (5 min each). Digital camera images and movies Digital camera images were acquired using an Olympus SP-350 8 Megapixels digital camera at the highest resolution mode. Digital movies were recoded using a QX5 Computer Microscope (Digital Blue Inc.). Cell counts and hyphal length Both biofilms and planktonic cultures were exposed to 20 mg/ml pronase in Tris buffer (10 mM Tris/HCl, pH 8.0, 2 mM EDTA) for 60 min to disperse cell aggregates according to a previously published protocol. [78] (Cell aggregates could not be dispersed sufficiently for either counting or hyphal length measurement by vortexing alone). Cells were counted in a hemacytometer. Hyphal length was measured from images acquired of dispersed cells using the Nikon/Metaview system described above.