Tween 20

ELISA plates were coated with whole-cell lysates from Bp or Bbss cultures, which had been sonicated and prepared as previously described [44 (link), 45 (link)]. Pooled plasma samples and serum samples from individual mice were diluted at 1:100 with sample buffer (PBS) containing 0.05% Tween 20 (AppliChem GmbH, Darmstadt, Germany) and 2% nonfat dry milk (Merck KGaA, Darmstadt, Germany). A computerized kinetic ELISA was applied as described previously [46 (link)]. Peroxidase-conjugated goat anti-mouse immunoglobulins (IgG, IgA, and IgM; MP Biomedicals, LLC, Heidelberg, Germany) at a dilution of 1:4000 (for Bp) and 1:3000 (for Bbss) served as a secondary detection antibody. Each test included negative and positive controls. All plasma and serum samples were tested in duplicate, and mean values are reported.

Standards of mycotoxins (AFB₁, ZEA and DON), carbonate–bicarbonate buffer glutaraldehyde solution (Grade II, 25%), glycine, tris (hydroxymethyl) amino-methane (ACS reagent, 99.8 + %) and sodium chloride (ACS reagent,  ≥ 99.0%) were purchased from Sigma–Aldrich (St. Louis, Missouri, USA). Skim milk (BD, Difco™ skim milk, Sparks, NV, USA), Tween 20 (molecular biology grade, Applichem, Darmstadt, Germany), SureBlue™ TMB Microwell peroxidase substrate (1-component) (KPL, Gaithersburg, MA, USA), sulfuric acid (Applichem, 95%–98% pure NF grade, Darmstadt, Germany), pyridine (Wako, Osaka, Japan), methanol (MeOH) (Merck, Darmstadt, Germany) and bovine serum albumin (BSA) (Fluka, St. Louis, USA) were purchased from the mentioned companies. The Micro BCA™ Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) and commercial ELISA Kit for AFB₁, ZEA and DON 2/3 (8335) (NEOGEN, Lansing, MI, USA) were used for protein and mycotoxin determination. Immunoaffinity columns for AFB₁ (NEOGEN, Glasgow, UK) and ZEA (R-BIOPHARM RHÔNE LTD, Glasgow, Scotland) were used to purify mycotoxin from a liquid solution. HPLC-grade acetonitrile (ACN), MeOH and water were purchased from J. T. Baker Inc. (Phillipsburg, NJ, USA). Phosphate-buffered saline (PBS) buffer was purchased from Biosesang Inc. (Seongnam, Republic of Korea).

For tissue analysis, samples were adjusted with LDS NuPAGE buffer (1×, Life Technologies, Darmstadt, Germany) and DTT (1 M, 10 % v/v) to obtain 20 µg protein/15 µl and incubated for 10 min at 70 °C. Samples were subjected to 4–12 % NuPAGE gradient gel (Life Technologies, Darmstadt, Germany) using MES buffer and a voltage of max. 150 V. For in vitro fibrillization analysis, peptide preparations were supplemented with LDS NuPAGE buffer (1×) and DTT (1 M, 10 % v/v) and boiled for 5 min at 95 °C. Samples were separated on 10 % SDS–polyacrylamide gels and blotted onto nitrocellulose membrane at 100 V for 2 h. Immunodetection of APP and A-beta was carried out by blocking the membranes for 1 h in blocking solution [0.2 % I-Block (Life Technologies, Darmstadt, Germany), 0.05 % Tween 20 (AppliChem, Darmstadt, Germany) in PBS] and incubation overnight at 4 °C with antibody 6E10 (Covance, Munich, Germany) at a dilution of 1:1000 in blocking solution. Blots were incubated with anti-mouse secondary antibody coupled with horseradish peroxidase (Thermo Scientific, Karlsruhe, Germany) and signals obtained by applying SuperSignal West Femto chemiluminescent substrate (Thermo Scientific, Karlsruhe, Germany) were detected using a CCD-camera imaging system (Stella Camera, Raytest, Straubenhardt, Germany).

The mice were sacrificed 2 or 3 weeks after laser injury by anaesthesia and cervical dislocation. The eyes were enucleated and adherent tissues removed. Fixation was done in 4% PFA (PBS) for one hour. Retinal and choroidal tissues were separated and washed in PBS (ICC-buffer) containing 1% BSA (PAA), 0.2% Tween20 (AppliChem) and 0.1% Triton-X 100 (Sigma). To visualize the endothelial cells (ECs) in neovascular areas, tissues were incubated in ICC-buffer with Alexa Fluor® 568-conjugated isolectin GS-IB4 (IB4) from Griffonia simplicifolia (life technologies) for 4h at 4°C. Then they were washed several times in ICC-buffer and mounted in Immumount on glass slides. The flatmounts were analyzed with an IX71 fluorescence microscope (Olympus) at 20-fold magnification. The size of IB4 positive CNV areas was quantified with CellF software (Olympus, Hamburg, Germany).

Cells grown on coverslips or slides containing spheroid sections were fixed with 4% paraformaldehyde (Merck, 1.04005) for 15 min at RT and then permeabilized with 0.3% Triton X-100 (Applichem, Darmstadt, Germany, A1388.0500) in PBS for 10 min at RT. Samples were blocked in PBS containing 15% FBS and 0.25% Tween-20 (Applichem, A4974.0500) for 1 h at RT. Next, samples were incubated with primary antibodies either for 1 h at RT or overnight at 4°C as indicated in Table S1. After several washes in blocking buffer, the corresponding secondary antibodies (Table S1) were added for 1 h at RT. Samples were counterstained and mounted with VectaShield containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories–H1200).

RNA from human cell lines and colonic tissues and tumors was isolated using Trizol reagent (Invitrogen) according to manufacturer guidelines. Tissues and tumor pieces were shredded using a homogenizer (T10 basic ULTRA-TURRAX). After reverse transcription (M-MuLV Reverse Transcriptase from NEB) of equal amounts of mRNA, quantitative real-time PCR analysis was performed using a qPCR MasterMix (72 mM Tris-HCl pH 8.8 (Roth), 19 mM (NH4)2SO4 (Roth), 0.01% Tween-20 (AppliChem), 3 mM MgCl₂, (Sigma-Aldrich), 1:80,000 SYBR Green (Invitrogen), 0.24 mM dNTPs, (dATP, dCTP, dGTP, dTTP, all dNTPs from Primetech), 19 U/ml Taq-polymerase (Primetech), 0.24% Triton X-100 (AppliChem), 300 mM Trehalose (Roth). Used primers are listed in Supplemental Table 1. For gene analysis, at least two different cDNAs (technical replicates) were used for qRT-PCR runs from one biological replicate. Biological replicates are independent experiments.