Lsm780 confocal laser scanning microscope

Cells were treated with purified collagen type II for 24 h, fixed with 4% paraformaldehyde, pretreated with 1% Triton X-100 and 0.5% bovine serum albumin (BSA) in phosphate buffer saline tween-20 (PBST), and blocked with 10% BSA in PBST for 30 min at room temperature. Cells were labeled with anti-SMAD1 antibody and treated with a secondary antibody, Alexa Fluor 555-conjugated goat anti-rabbit IgG, and then stained with DAPI. Signals were captured with a Zeiss LSM780 confocal laser scanning microscope (Zeiss, Jena, Germany).

To prepare the frozen sections, LGs were fixed with 2% paraformaldehyde in PBS (pH 7.4) for 20 to 30 minutes, frozen in 2-methylbutane (isopentane, #M32631-500ML; Sigma-Aldrich) cooled by liquid nitrogen, and 10-µm sections were prepared using a Bright OTF 5000-LS004 Cryostat (Hacker Instruments, Winnsboro, SC, USA). Sections were blocked with 5% goat serum in Tris-buffered saline containing 0.1% Tween 20 (TBST). The following primary antibody was used for immunostaining: heparan sulfate proteoglycan (clone A7L6 #MAB1948P, RRID:AB_10615958; Sigma-Aldrich).
Appropriate Invitrogen secondary antibodies were obtained from Thermo Fisher Scientific. Images were taken using a Zeiss LSM 780 confocal laser scanning microscope (RRID:SCR_020922; Carl Zeiss Microscopy, Jena, Germany). Isotype-specific immunoglobulins (normal rabbit IgG, #12-370, RRID:AB_145841; normal mouse IgG, #12-371, RRID:AB_145840; Sigma-Aldrich) or pre-immune serum, as a substitute for the primary antibody, were used for negative controls.

Cultures of WT, Δbac, ΔddE, ΔddF, and ΔddF-Comp strains were treated with the LIVE/DEAD™ BacLight™ Bacterial Viability Kit (Thermo Fisher Scientific, Landsmeer Netherlands) to analyze the viability of the bacteria at 24 h of growth. The staining procedure was carried out following the manufacturer’s instructions. Stained bacterial solutions were imaged with a ZEISS LSM 780 confocal laser scanning microscope equipped with a 40x/1.3 oil immersion objective (Carl Zeiss Micro Imaging GmbH, München, Germany). The SYTO 9 dye was excited with a laser at 488 nm and detected between 493 and 560 nm; and the propidium iodide dye was excited at 561 nm and detected between 584 and 718 nm. The images were acquired with the Zen software (Carl Zeiss Micro Imaging GmbH, Germany), and analyzed with the ImageJ software (National Institute of Health, Bethesda, MD, USA).

For all microscopy experiments, MM170 cells were cultured on 4-well Nunc Lab-Tek (Nunc, Germany) chamber slides and allowed to adhere for 24 h before imaging. DIC live cell imaging was performed on a Zeiss spinning disk confocal microscope (Zeiss, Oberkochen, Germany) using a 63× oil immersion objective in a 37 °C/5% CO₂ atmosphere. Cells were imaged in growth media in the presence of 1.25 μM NaD1 at specified time points over 24 h. Image processing and data analysis were performed using the ZEN imaging software (Zeiss). Fluorescence live cell imaging was performed on a Zeiss LSM-780 Confocal Laser Scanning Microscope (Zeiss) using a ×63 oil immersion objective in a 37 °C/5% CO₂ atmosphere. Cells were preincubated with mitochondrial membrane potential-dependent mitochondrial stain, MTR (Thermo Fisher) at 50 nM in the growth medium for 15 min at 37 °C before imaging. Cells were then imaged in media containing 4 kDa FITC-dextran at 10 μg/ml, in the presence of 1.25 μM NaD1 over 4 h at 30 s intervals. Image processing and data analysis were performed using the ZEN software (Zeiss). Kinetic analysis of FITC-dextran uptake versus loss in MTR fluorescence was performed in Excel with mean fluorescence intensities in individual cells captured across multiple imaging sessions and displayed as average across all cells.

ARPE-19 cells were seeded and cultured on 8-well chamber slides (Thermo Fisher Scientific, Rochester, NY, USA). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, blocked with 1% bovine serum albumin (Sigma) for 1 h at room temperature, and then incubated with FITC-conjugated anti-α-SMA antibody (1:100) overnight at 4°C. After being washed with phosphate-buffered saline, the nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Life Technologies). Then the cells were mounted with Fluorescent Mounting Medium (Dako, Carpinteria, CA, USA) and examined using a Zeiss LSM 780 confocal laser scanning microscope (Carl Zeiss, Germany).

The coding sequence of Sl3-MMP was amplified from pMD19-Sl3-MMP using a pair of primers Sl3-MMP-GFP-F and Sl3-MMP-GFP-R (Additional file 3) and inserted into pFGC-Egfp at BamHI/XbaI sites. The recombinant plasmid pFGC-Sl3-MMP and the empty vector pFGC-Egfp were introduced into onion epidermal cells by particle bombardment method. Particle bombardment was performed with a PDS-1000 (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. GFP was detected 24 h after bombardment. Plasmolysis was achieved by treating the bombarded onion epidermal cells with 0.8 M mannitol for 10 min. Microscopic observation was performed using a Zeiss LSM 780 confocal laser scanning microscope (Carl Zeiss, Germany) and representative photographs were taken.