Lsm 5 pascal laser scanning microscope

Biofilms of H. pylori G27 and mutants were prepared as described above using BB10 media, however, for confocal laser-scanning microscopy (CLSM), μ-Slide 8-well glass bottom chamber slides (ibidi, Germany) were used instead of 96-well microtiter plates. Three-day-old biofilms were stained with FM^®1–43 (Invitrogen) or FilmTracer LIVE/DEAD biofilm viability kit (Invitrogen) according to the manufacturer’s instructions. Stained biofilms were visualized by CLSM with an LSM 5 Pascal laser-scanning microscope (Zeiss) and images were acquired using Imaris software (Bitplane). Biomass analysis of biofilm was carried out using FM^®1–43 stained z-stack images (0.15 μm thickness) obtained by CLSM from randomly selected areas. The biomass of biofilms was determined using COMSTAT (27).

HCC cells were fixed with 3% paraformaldehyde and permeabilized using 0.2% Triton X-100. Then the fixed cells were incubated with the YAP (1:500) primary antibody. The secondary antibody is an Alexa Fluor–conjugated IgG (Invitrogen, Carlsbad, CA, USA). Fluorescence confocal images were captured using a LSM 5 Pascal Laser Scanning Microscope (Zeiss Germany, Oberkochen, Germany) using a × 40 lens and Laser Scanning Microscope LSM PASCAL software (version 4.2 SP1).

Confocal fluorescence images of phyB-GFP were taken from the epidermis and the first subepidermal layers of the upper third portion of the hypocotyl. We used an LSM5 Pascal laser scanning microscope (Zeiss) with a water-immersion objective lens (C-Apochromat 40×/1.2; Zeiss). Probes were excited with an argon laser (λ = 488 nm), and fluorescence was detected using a BP 505-530 filter. Images were taken from individual nuclei, and image analysis was performed as in (5 (link)). Each data point consists of two replicates coming from two independent experiments showing the same trend. Replicates consisted of the average of three plants coming from the same plate, and three nuclei per plant were analyzed.

Cells grown on glass coverslips were washed with PBS, fixed for 15 min with 4% formaldehyde, and washed with PBS. After quenching residual formaldehyde with 0.1 M glycine, cells were washed with PBS, permeabilized for 10 min at room temperature with 0.1% Triton X-100, and then washed with PBS. After blocking in 5% fetal bovine serum, cells sequentially were incubated with primary and secondary antibodies diluted in 1% bovine serum albumin (BSA). Apart from the calnexin antibody, which was incubated overnight at 4°C, primary antibodies were incubated for 1 h at room temperature and secondary antibodies were incubated for 45 min at room temperature. Cells were washed in PBS after each antibody incubation. Coverslips were mounted in ProLong Gold antifade reagent (Invitrogen) and analyzed using an LSM 5 PASCAL laser scanning microscope (Carl Zeiss).
The following primary antibodies were used at the indicated dilution: mouse anti-Myc tag (05-724; 1/500; Millipore), rabbit anti-calnexin (2679S; 1/50; New England BioLabs), rabbit anti-Giantin (ab80864; 1/500; Abcam), mouse anti-GM130 (610822; 1/500; BD Pharmingen), sheep anti-TGN46 (AHP500GT; 1/500; AbD Serotec), and rabbit anti-HRV16 2C (this study; 1/500). Secondary antibodies were obtained from Jackson ImmunoResearch (1/200).

Biofilms of H. pylori G27 WT were prepared as described above using BB10 media. To use confocal laser scanning microscopy (CLSM), μ-Slide 8-well glass bottom chamber slides (ibidi, Germany) were used in place of 96-well microtiter plates. Growth and treatment were the same for other antibiotic exposure experiments. After 3 days, biofilms were stained using a FilmTracer™ FM^®1–43 (Invitrogen) or FilmTracer™ LIVE/DEAD Biofilm Viability Kit (Invitrogen) according to the manufacturer’s instructions. Stained biofilms were visualized by CLSM with an LSM 5 Pascal Laser Scanning Microscope (Zeiss), and images were acquired using Imaris software (Bitplane). Biomass analysis of biofilm was carried out using FM^®1–43 stained z-stack images (0.15 μm thickness) obtained by CLSM from randomly selected areas. The biomass of biofilms was determined using COMSTAT [49 (link)].

GC cells that were transfected with corresponding miRNA vectors were seeded on chamber slides and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Then, cells were incubated with primary antibodies including E-cadherin (Abcam) and Vimentin (Abcam) at 4°C overnight. Then, the slides were incubated with matched secondary antibodies (Invitrogen) at room temperature for 1 hour. The nuclear of EC cells were stained with DAPI (Sigma) at room temperature for 10 minutes. Fluorescence confocal images were captured using a LSM 5 Pascal Laser Scanning Microscope (Zeiss Germany, Oberkochen, Germany).

GC cells were seeded on chamber slides and fixed with 4% paraformaldehyde for 10 min at room temperature. Then, cells were incubated with antibodies against E‐cadherin (Abcam), N‐cadherin (Abcam) or vimentin (Abcam) at 4 °C overnight. Then, the slides were incubated with matched secondary antibodies (Invitrogen/Thermo Fisher Scientific) at room temperature for 1 h. The nuclei of GC cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma‐Aldrich) at room temperature for 10 min. Fluorescence confocal images were captured using an LSM 5 Pascal laser scanning microscope (Zeiss, Oberkochen, Germany).