Axiocam 105 color camera

Saccharomyces cerevisiae strains were pre-cultured in YPD at 24 ± 2°C for 24 h. Cultures underwent serial dilution in 0.85% sterile saline and were inoculated to a 10⁻⁵ concentration on SLAD agar plates. For experimental conditions, SLAD was supplemented with 100 µM or 200 µM exogenous 2-PE. Plates were sealed with parafilm and incubated inverted for three days at 30°C. Following incubation, 10–15 colonies were randomly selected and photographed from each dish at 2.5 X total magnification using a ZEISS Axio Lab.A1 FL-LED microscope equipped with a ZEISS Axiocam 105 color camera (2.2 μm pixel resolution) and saved as a JPEG image. The assay was carried out in triplicate per condition, whereafter, ten whole colony photographs from each assay condition (n = 30) were chosen randomly selected for further image analysis and processing (n = 540).

Five micrometer thick paraffin-embedded tissue sections were deparaffinized and rehydrated using Xylene and a descending ethanol gradient (100%, 95%, 70%, pure dH₂O). Three percent acetic acid was applied to the slides for 3 min. Alcian blue solution (pH 2.5) was added to slides for 30 min to stain the goblet cells. Three percent acetic acid was applied to the slides for ~30 s to remove excess Alcian blue staining. Slides were rinsed in running tap water for 5 min followed by two changes of distilled water. Nuclear Fast Red solution was applied for 5 min to stain nuclei. Slides were rinsed with running tap water for 2 min, transferred to two changes of distilled water, and then dehydrated with an ascending ethanol gradient (70, 95, and 100%). Slides were mounted. Images were acquired using a Zeiss Observer D1 Inverted Phase Contrast Fluorescence Microscope, Axiocam 105 color camera, and Zeiss Zen pro microscope software (Stony Brook University).

Cells were serum-starved for 24 hr in medium containing 0.1% FBS. Cell culture inserts for 24-well plates containing polyethylene terephthalate membranes with 8 μm pores (Corning Inc., Corning, NY, USA) were coated with 5 μg/mL human plasma fibronectin purified protein (EMD Millipore Corporation). Cells were seeded at a density of 315,000 cells per insert in 250 μL medium containing 0.1% FBS and inserts were immersed in 500 μL medium containing 10% FBS supplemented with 10 ng/mL PDGF-AA for 4 hr. Migrated cells were subsequently fixed in 4% PFA in PBS for 10 min and stained in 0.1% crystal violet in 10% ethanol for 10 min. Dried inserts were photographed using an Axiocam 105 color camera fitted onto a Stemi 508 stereo microscope (Carl Zeiss Microscopy, LLC). Five fields of cells from each of three independent trials were photographed and quantified by measuring integrated density with ImageJ software (version 1.50i; National Institutes of Health, Bethesda, MA, USA). Statistical analyses were performed with Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA) using a two-tailed unpaired t-test.

P. aeruginosa strain PAO1 was inoculated from a streak plate into 5 ml LB and incubated overnight at 37°C with shaking at 220 rpm. We inoculated 1.98 ml of SCFM1 or PGM-SCFM1 (pH 7.0) with 20 μl of P. aeruginosa LB broth culture (OD₆₀₀ of 0.05; ∼10⁶ CFU/ml final concentration) in a polystyrene 24-well plate (88 (link)). Both media were evaluated in one 24-well plate (4 control wells and 4 growth wells per medium). The plate was covered and incubated statically at 37°C for 72 h. After incubation, gross phenotypes of P. aeruginosa growth (Fig. S8) were photographed using a top-down view with a Stemi 508 stereo microscope with an Axiocam 105 color camera (Zeiss). Following visualization, aggregates were mechanically disrupted by pipetting using wide-bore pipette tips (USA Scientific). Disrupted samples were aliquoted for measurements of growth (CFU per milliliter and pH; Fig. S8) and metabolomics analysis. All four biological replicates from each culture condition were serially diluted, spotted onto LB agar, incubated at 37°C overnight, and counted to determine CFU per milliliter.

IHC staining of o.t. CDX and metastases thereof were conducted using anti-CLA (clone HECA-452, Miltenyi Biotec, 130-091-634, 1/100), anti-CD66c (polyclonal, Abcam, ab199277, 1/100), anti-CD318 (polyclonal, Invitrogen, PA5-17245, 1/50), anti-TSPAN8 (polyclonal, Abcam, ab230488, 1/100), anti-rabbit horseradish-peroxidase (HRP) (polyclonal, Medac, 414142F), and Avidin-HRP (Invitrogen 18-4100-94). Sections were boiled for 20 min in Target Retrieval Solution (Agilent) either with pH 6.0 for CLA, CD66c, and TSPAN8 or pH 9.0 for CD318 with subsequent incubation for 5 min in ice cold water and rinsing twice with TBS buffer for 5 min each. Afterwards, slices were incubated for 10 min with 3% hydrogen peroxide at RT followed by two 5 min washing steps with TBS. Unspecific antibody binding was averted by a 20 min blocking step with Fish block (SurModics). Primary antibody staining was performed overnight at 4 °C. Samples were washed and secondary antibody (anti-rabbit-HRP, undiluted or Avidin-HRP, 1:500) was added for 30 min (anti-rabbit-HRP) or 1 h (Avidin-HRP) at RT. Subsequently, samples were washed and enzyme substrate (AEC, BD Pharmingen, Cat. 551015) was added to the sections for 20 min at RT. Images were acquired using an Axioskop II microscope (Zeiss) and an Axiocam 105 color camera (Zeiss).

Spines were decalcified in 20% ethylenediaminetetracetic acid (EDTA) at 4 °C for 15 days before embedding in paraffin and 7-μm mid-coronal sections were prepared. Xylene deparaffinization followed by graded ethanol rehydration preceded all staining protocols. Safranin O/Fast Green/Hematoxylin-stained slides were imaged using Axio Imager 2 microscope, 5×/0.15 N-Achroplan or 10×/0.3 EC Plan-Neofluar objectives, Axiocam 105 color camera, and Zen2TM software (Carl Zeiss). Scoring was performed using a modified Thompson grading scale (S1) by 5–7 blinded observers^{70 (link),71 (link)}. Endplate scoring was performed by five blinded observers following Boos criterion^{37 (link)}. Ten mice per genotype with three discs per mouse in both caudal and lumbar levels were used.