Lsm510 axiovert 100 m

For confocal microscopy biofilms were formed on Nunc Lab-Tek 8-well chamber slide (No. 155411) containing a borosilicate glass base 100 μm thick. Overnight cultures of K279a and F60 were standardized to contain approximately 10⁶ CFU/ml and for each test condition (presence or absence of 200 μM Dip), chambers were filled with 400 μl of the standardized inoculum. After 48 h of incubation, the wells were rinsed with sterile physiological saline (0.9% NaCl) in order to eliminate any non-adherent bacteria. The wells were refilled with physiological saline containing 2.5 μM Syto9® (Molecular Probes, Grand Island, NY), a green fluorescent nucleic acid marker, and incubated in the dark for 20 min. Images were acquired with a confocal laser-scanning microscope Carl Zeiss LSM510-Axiovert 100 M by sequentially scanning with a 488 nm argon laser using a 40X water immersion objective lens, at the Instituto de Investigaciones Biomédicas (Pontificia Universidad Católica Argentina-CONICET, Argentina). Emitted fluorescence was recorded within the 505–530 nm range to visualize Syto9 fluorescence, and Z-stacks were captured every10 μm at different areas in the well. Images were analyzed using the ZEN 2009 Light Edition (Carl Zeiss) and three-dimensional projections of the biofilms' structure were reconstructed using the ImageJ program (ImageJ. Available online: http://rsbweb.nih.gov/ij/).

Samples from the cucumber collar-region of CGMMV-infected plants (with or without P. spinosum), and from healthy control plants, collected on 13 dpvi, were sliced and fixed with 4% formaldehyde and 0.2% glutaraldehyde, as described previously (Reingold et al., 2015 (link)). The slices were washed twice with PBS-T (phosphate-buffered saline with 0.05% Tween-20), blocked using PBS with 1% milk (0% fat) for 30 min and incubated overnight at 4°C with IgG antibodies specific for CGMMV (Antignus et al., 1990 (link); Antignus et al., 2001 (link)). Slices were washed twice with PBS-T, and the secondary antibody, goat anti-rabbit IgG-conjugated Alexa Fluor 488 (Invitrogen), was added to the slices in a 1:1,000 dilution in PBS followed by incubation at 37°C for 3 h. The slices were washed twice with PBS-T and kept in PBS in a sealed box. The fluorescence signal of the slices was detected using confocal microscopy (LSM510 Axiovert 100 M, ZEISS, USA).

Cellular cortactin expression was visualized by immunofluorescence microscopy (Zeiss LSM 510 Axiovert 100 M, Zeiss, Thornwood, NY). Briefly, cells were cultured to confluence on glass cover slips and fixed in 4% paraformaldehyde in PBS. The samples were rinsed three times, permeabilized with 1.2% Triton X-100 for 5 min, rinsed three times and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h before staining with 1:100 cortactin primary antibody (Cell Signaling Technology, Danvers, MA) followed by Alexa Fluor-conjugated secondary antibody (Invitrogen, Carlsbad, CA). The stained cells were mounted with ProLong® gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA) and visualized by fluorescence microscopy. All microscopic exposure conditions were set the same between samples for fluorescence intensity comparison.