Lsm 700 inverted confocal laser scanning microscope

UGT79B31 was amplified with primers, GFP-79B31f/GFP-79B31r and 79B31-GFPf/79B31-GFPr (Table S1) for fusion to N- and C-terminal GFP, respectively; the amplification products were cloned into the pGWB5 or pGWB6 vectors (Nakagawa et al. 2007 (link)) using the Gateway Cloning System. The resultant plasmids pUGT79B31-GFP (UGT79B31 in pGWB5) and pGFP-UGT79B31 (UGT79B31 in pGWB6) were used to stably transform Agrobacterium tumefaciens strain GV3101 and transiently transform N. benthamiana leaves by the agroinfiltration method (Leuzinger et al. 2013 ). Plasmid pBIN61-P19 was used for the control (http://www.plantsci.cam.ac.uk/research/davidbaulcombe/methods/protocols/pbin61-p19.doc/view). Three days after infiltration, leaves were analyzed using a Zeiss LSM700 inverted confocal laser scanning microscope with a 40× dry objective (Zeiss). A diode laser with 488 nm excitation and appropriate filters (505–600 nm) were used to detect GFP fluorescence. ZEN 2011 software (Zeiss) was used for data analysis.

Indirect immunofluorescence analyses were carried out following the methods described by Inbar et al. [34 (link)]. Briefly, late log phase promastigotes were washed twice in PBS and then fixed in 1% formaldehyde/PBS on a slide for 10 min before permeabilization by exposure to 0.2% TritonX-100/PBS (PBST) for 10 min. Cells were incubated with blocking solution [5% (v/v) non-fat dried skimmed milk powder/PBST] for 30 min at room temperature, incubated with rabbit anti-PKAR3 (this study), rabbit anti-promastigote membrane proteins [28 (link)] or mice anti-HA tag antibodies (1:500 dilution) for one hour. Subsequently, slides were washed three times with PBS-Tween and incubated with the fluorescent secondary antibody in darkness for 30 min. Slides were washed three times with PBS-Tween and a drop of DAPI Fluoromount G (Southern Biotech) was added. Slides were covered with slips, sealed, and then kept in darkness. Subsequently, they were examined using a Zeiss LSM 700 inverted confocal laser scanning microscope. Image processing was done using Zen lite software, Zeiss.

CLSM was used to confirm the effect of peptide 8 on mature biofilm respect the controls. MSRP were grown in chambered cover glass (μ Slide 4 well; ibidi GmbH, Germany) in a static condition for 24 h. Peptide 8 was added on a 1-day-old biofilm at 12.5 μM. Bacterial suspensions incubated with medium alone were used as a positive control. After 24 h, biofilms were rinsed with PBS and stained by using a LIVE/DEAD^® BacLight Bacteria Viability stains (Life Technologies, Italy). After the staining, the images were observed using a LSM 700 inverted confocal laser-scanning microscope (Zeiss, Italy). The areas were scanned using a 10X objective lens with the signal recorded in the green channel for Syto9 (excitation 488 nm, emission 500–525 nm) and in red channel for PI (excitation 500–550 nm, emission 610–650 nm).