Bovine serum albumin fraction 5

Protein localization was assessed using standard immunocytochemical procedures as described previously [57 (link)]. Formalin-fixed cell smears were prepared and boiled in a microwave oven twice (2 × 10 min, 200 W, water bath) in citrate buffer (pH 6.0, 0.1 mM citric acid solution, 0.1 mM sodium citrate solution; Avantor Performance Materials; Poznan, Poland). After each step of microwaving antigen retrieval, slides were chilled (20 min, room temperature) and incubated for 3 min in a 3% hydrogen peroxide solution (Avantor Performance Materials, Poznan, Poland). The next step included placing the slides in TBS-T buffer (containing 3% bovine serum albumin, fraction V; Merck KGaA; Darmstadt, Germany) for 1 h at RT. The same primary antibodies were used as described in the western blot section at a 1:100 dilution for all immunocytochemical reactions. Unbounded antibodies were washed in TBS-T buffer (3 × , 10 min, RT). The antigens were visualized using EnVision + /HRP system and 3,3′-diaminobenzidine chromogen (Agilent Dako, Santa Clara, USA). Controls included detection reactions carried out under identical conditions, except that the primary antibodies were replaced by nonimmune serum. The slides were assessed using light microscopy (Olympus CX41 microscope, Olympus Corporation, Tokyo, Japan).

Cells were washed with warm (37°C) phosphate buffered saline (PBS, Wisent Inc.) and fixed with 4% paraformaldehyde at room temperature for 10 minutes. The cell membranes were permeabilized with 0.5% v/v Triton-X100 (Sigma) in PBS. Antigens were blocked with 1% bovine serum albumin fraction V (Merck) in PBS for 1 hr at room temperature followed by immunolabeling. The primary and secondary antibodies used, incubation protocol, and DNA staining and are described in Supplementary Methods S1. An Olympus 1X81 spinning disc confocal microscope (Olympus Canada Inc., Richmond Hill, ON, Canada) with Yokogawa scan head (Yokogawa Corporation of America, Sugar Land, TX, USA) was used to visualize the cells with 20X air or 40X water immersion lenses (Carl Zeiss AG, Germany). Images were processed using Volocity 5.5 imaging software.

All chemicals used in this work were analytical grade and were used as received. EDTA and tween-20 were purchased from Sigma-Aldrich. Bovine serum albumin, fraction V, (BSA) was from Merck. The Cy5-streptavidin was purchased from Abcam US (United States). Phosphate buffer saline (1× PBS, 1.8 mM Na₂HPO₄·7H₂O, 10 mM KH₂PO₄, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) was used for all solution preparations. 1× PBS was used as working buffer, PBS containing 0.05% tween-20 was used as a washing buffer, and PBS containing 0.05% tween-20 and 2% (w/v) BSA was used as a blocking buffer. All solutions and buffers were prepared using sterile Type 1 pure water from Merck Millipore.
The double-biotin DNA linkage, 24 base poly-T oligonucleotide with the 3′ and 5′ biotin-modified DNA, was purchased from Integrated DNA Technologies (USA). Goat anti-E. coli O157:H7 (#5310-0326), biotin-labelled goat anti-E. coli O157:H7 (#16-95-90), goat anti-Salmonella CSA-1 (#5310-0322), and biotin-labelled goat anti-Salmonella CSA-1 (#5360-0031) were purchased from KPL BacTrace® US (United States).

Protein concentration was quantified using the Bradford method 15. One hundred microliters of sample was added to 2 mL of Bradford reagent [100 mg Coomassie G250 (Sigma‐Aldrich), 50 mL of 95% ethanol (Sigma‐Aldrich), 100 mL of phosphoric acid 85% (Sigma‐Aldrich), and water to 1 L]. The mixture was incubated for 10 min at room temperature. The absorbance was read at 595 nm. Bovine serum albumin fraction V (Merck) was used as a reference standard.

Gelatin (denatured collagen I, from bovine skin), vitronectin (from human plasma), bovine serum albumin (BSA, fraction V) and Efavirenz were obtained from Sigma-Aldrich (Milan, Italy). Human recombinant IL-1β, TNF-α, and IFN-γ, and fibronectin from human plasma were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). MAbs directed against the α5β1, αvβ3, αvβ5, or α6β4 integrins were obtained from Chemicon-Merck-Millipore (Darmstadt, Germany). The anti-CD31 (PECAM-1) mAb was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The Tat peptides (11–25), (46–60), and (66–80) were from UFP Service, University of Ferrara, Italy. Phosphate-buffered saline (PBS) solution, cell growth medium (RPMI 1640) and media supplements were obtained from Invitrogen-Life Technologies (Milan, Italy). Fetal bovine serum (FBS) was from HyClone (Logan, UT, USA).