Lsm 700 laser scanning

Bacterial biofilm formation assays were conducted following an established method from our previous study[25]. Briefly, the printed and UV-sterilized devices (cuboids, tablets or finger implants) with the developed formulations were inoculated with bacteria (P. aeruginosa tagged with the fluorescent protein mCherry, OD₆₀₀ = 0.01) and incubated at 37°C with shaking at 60 rpm shaking for 72 h in RPMI-1640. Samples were washed 3 times with 60 rpm shaking in PBS to remove planktonic and weakly adhered bacteria before being allowed to air dry. Samples were examined using a Carl Zeiss LSM 700 laser scanning confocal microscope fitted with 405 nm, 488 nm and 555 nm excitation lasers and a 10X/NA 0.3 objective. Images were acquired using ZEN 2009 imaging software (Carl Zeiss). Bacterial surface coverage was quantified using ImageJ 1.44 software (National Institutes of Health, USA) and Comstat 2^{[44 (link)]}.

Zeiss LSM 700 laser scanning confocal microscope was used to acquire images of antibody-stained retinas with EC Plan-Neofluar × 40 or × 60 oil objectives. Images were processed using Imaris (Bitplane) or ImageJ 1.47u (NIH).

HeLa cells expressing GP or HeLa cells expressing thermolysin-treated GP (RBD-exposed cells) were seeded on cover slips. The cells were treated with 30 µg/mL of R9-HuscFvs/HuscFvs and incubated at 37 °C for 2 h to allow cell adherence. The cells were then washed, fixed with 4% paraformaldehyde in PBS for 30 min, blocked with 5% BSA in PBS for 1 h, and stained with anti-6× His (1:200 dilution) and anti-GP antibodies (1:500 dilution) for 1 h. The cells were then stained with fluorophore-conjugated anti-isotype antibody (1:300 dilution) for 1 h and counterstained with DAPI (1:5000 dilution) for 15 min at 4 °C. The stained cells were mounted onto slides, sealed with nail polish, and subjected to sectional confocal microscopy by using Carl Zeiss LSM 700 laser scanning.
For flow cytometric analysis of the binding of R9-HuscFvs/HuscFvs to the RBD, HeLa cells expressing GP or HeLa cells were treated with thermolysin, as described above. The cells were incubated with 1 µg/mL of the antibodies in FACS buffer (2% FBS and 0.02% sodium azide in PBS) on ice for 1 h. The cells were washed and stained with anti-6× His (1:200 dilution) at room temperature for 1 h, followed by the fluorophore-conjugated secondary antibody (1:300 dilution) on ice for 1 h. The stained cells were fixed with 1% paraformaldehyde in PBS and analyzed by using the BD LSR Fortessa flow cytometer.

Confocal microscopy was performed using a Zeiss LSM700 laser scanning confocal microscope. The SK-HEP-1 cells (ATCC) or 293 cells (Life technologies) were seeded on a 35-mm glass-bottom dish (D35-20-1-N, In Vitro Scientific) and transfected with the indicated plasmids for 48 h. 5 μM CnB-GFP or rhodamine-conjugated CnB was then added to the cells and incubated for 30 min, followed by three washes with PBS or acid-stripping buffer. The localization of the fluorescence was determined.