Lsm700 axioobserver

MDCK cells were grown on 12 mm Transwell polycarbonate membranes (0.4 μm pore; Sigma) and transfected with the Lucy-Flag-Olfr1393 construct that was described previously57 (link) along with RTP1S59 (link) (modified from RTP1L). The cleavable Lucy tag was used to promote functional expression of ORs in heterologous cells. After 24 h, the media was replaced and the cells were allowed to grow to confluency and polarize (72 h). To label the apical membrane, the top side of the Transwell membranes containing live, non-permeabilized cells was exposed to a rabbit polyclonal anti-Flag antibody (Sigma) at 4 °C. Subsequently, the cells were washed, fixed with 4% paraformaldehyde, permeabilized (0.3% Triton X-100) and exposed to a mouse gp135 antibody26 . To detect total Flag-Olfr1393, the live cell labeling steps were skipped and the cells were immediately fixed and permeabilized and probed for Flag and gp135. The confocal images were taken with a Zeiss AxioObserver LSM700 and Zen software.

Sections were analyzed and images were acquired using a Zeiss LSMDUO confocal laser scanning microscope with a META module (Carl Zeiss MicroImaging GmbH, Germany) microscope, the LSM700 AxioObserver. The Zen 2011 (LSM 700, Zeiss software, Oberkochen, Germany) built-in “colocalization view” was used to highlight the expression of both antibodies’ signals [76 (link),77 (link),78 (link)], in order to produce a “colocalization” signal, along with the display profile, the scatter plot and fluorescent signal measurements. Each image was rapidly acquired to minimize photodegradation.

GUVs were imaged with a laser scanning confocal microscope (LSM 700 AxioObserver, Zeiss). The microscope was equipped with a Plan-Apochromat 63x/1.40 Oil DIC M27 (Zeiss). The LSM 700 operates with solid-lasers (polarization-preserving single-mode fibers) at a wavelength of 639 nm and 488 nm. Signals were detected after appropriate filtering on a photomultiplier. Typically a z-stack was performed with a step size of 500 nm. Detector amplification, laser power and pinhole were kept constant for all measurements. Images were analyzed by thresholding the C-Bodipy signal; the average pixel value of signals underneath the obtained mask, which essentially follows the GUV surface, was calculated in both the C-Bodipy and the apoA-I-Alexa647 channel (see Fig. S1).

All images were acquired with an LSM700 AxioObserver laser scanning confocal microscope equipped with a plan Apochromat 40x/1.3 Oil DIC M27 (Zeiss, Oberkochen, Germany) objective, using a gallium arsenide phosphide photomultiplier tube (GaAsp-PMT) detector controlled by Zen black software (version 8.0.7.273, Zeiss, Oberkochen, Germany). After acquisition, images were processed using the Fiji ImageJ2 software (National Institutes of Health, Bethesda, MD, USA).

Biofilms were grown on
glass coverslips (13
mm Ø, no. 1.5 thickness) in a rolling biofilm bioreactor system^{48 (link)} (20 rpm) in FAB 10 mM glucose medium, inoculated
with diluted (OD600 nm = 0.01) bacteria from overnight cultures in
LB. For the biofilm samples that were treated with 6f, a concentration of 10 μM was supplemented to the bioreactor’
media at the start of the experiments. The biofilms were cultivated
at 30 °C for 24 h, then washed in PBS to remove loosely attached
cells and incubated for a further 6 or 24 h in fresh medium supplemented
with various treatments. These included free ciprofloxacin 60 μg/mL
(× 300 the MIC of planktonic P. aeruginosa cells,^{44 (link)}6f at 20 μM and ciprofloxacin
in combination with 6f. Biofilms exposed to each treatment
were washed in PBS, and the viability of attached cells was evaluated
by fluorescent staining using the LIVE/DEAD BacLight bacterial viability
kit (Molecular Probes, Life Technologies) according to manufacturer
instructions. Following staining, coverslips were rinsed with distilled
water and imaged using a LSM700 AxioObserver (Carl Zeiss, Germany)
confocal laser scanning microscope (CLSM). Viable and nonviable biofilm
biomass quantification from image stacks of biofilms was done with
Fiji-ImageJ software. Live/dead ratios were established for each treatment
and compared to untreated controls.

Z-stacks were captured at 1440 × 1440 pixels, 16-bit and averaged X2 using a Plan-Apochromat 63X/1.4 oil DIC M27 objective and 1.0 zoom to produce 101.54 × 101.54 µm uncompressed images from the most dorsal CA1. Two stitched Z-stacks were set to be captured at 1 air unit (0.8 µm slicing) to 568 nm wavelength using a Laser Scanning Confocal Microscope LSM700 AxioObserver (Carl Zeiss AG, Oberkochen, Germany). Protocol specifications were followed in all repeats of each experiment regardless of age of the animal or species; thus, pinhole, gain and offset configurations were reused in all experiments. Detection wavelengths were 300–483 nm for DAPI and 560–600 nm for Alexa 568 (Iba1). Uncompressed czi format was used for processing. Representative 3D reconstructions for all 6 groups at P10–P11 and P18–P19 are shown in Figure 2.