Alexa fluor 488 conjugated anti mouse igg

β-catenin and E-cadherin staining were performed on methanol fixed submerged cultures. Primary antibody against total β-catenin (Cell Signaling) and E-cadherin (Invitrogen) were used followed by an Alexa Fluor 568-conjugated anti-rabbit IgG or an Alexa Fluor 488-conjugated anti-mouse IgG (both from Life Technologies). Nuclei were stained with DAPI and samples were mounted with Prolong Gold Antifade reagent (Life Technologies). Images were taken using Axiovert 200 M microscope and analyzed with Axiovision 4.5 software (Carl Zeiss; Marly Le Roi, France).

A549 cells were dissociated by dissociation solution (Millipore) and filtered through 40 μm strainer and centrifuged at 1500 rpm for 3 min. Thirty-four million cells were incubated with 70 μg of SP1-B7 for 30 min at RT and washed by PBS (pH 7.4) including 5% FBS and further incubated with Alexa fluor 488-conjugated anti-mouse IgG (Life technologies) for 30 min at RT in the dark. After washes, the cells were isolated by BD FACSAria (BD Biosciences). After cell sorting, SP1-B7-positive or -negative cells were counted with 0.4% Trypan blue (Welgene) and live cells were used for the clonogenic survival assay.

Frozen sections of colorectal cancer tissues (n = 10) and background normal tissues (n = 10) were selected from the tissue bank and cut at a thickness of 6 μm using a cryostat. The sections were fixed with 4% paraformaldehyde in PBS at room temperature for 30min. Sections were incubated for 30 min in 5% BSA blocking solution and probed with monoclonal mouse anti-human NHERF1 primary antibody (1:100) (BD Biosciences) and polyclonal rabbit anti-human VEGFR2 primary antibody (1:100) (Santa-Cruz). This study employed controls that omitted the primary and secondary antibodies. Following extensive washings, sections were incubated with an Alexa Fluor^® 488-conjugated anti-mouse IgG (Life Technologies, Massachusetts, USA) at 1:200 dilution and Alexa Fluor^® 594-conjugated anti-rabbit IgG (Life Technologies) at 1:200 dilution in the dark for 1 h. After washing three times to remove the unbound secondary antibody, cell nuclei were stained with Hoechst 33258 (Sigma). The sections were finally mounted with FluorSave™ (Calbiochem-Novabiochem Ltd., Nottingham, UK) and visualized with a confocal microscope (Leica Microsystems LAS AF-TCS SP5. Wetzlar, Germany).

The paraffin blocks of fixed lung tissue were sectioned (4 mm) onto microscope slides and incubated at 60 °C for 30 min. The slides were then immersed in xylene twice, and in 100% and 70% ethanol. After removing all remaining paraffin, the slides were rinsed with tap water and distilled water. To enhance the staining intensity, the washed slides were heated in pH 6 citrate buffer at 118 °C for 20 min. The slides were blocked with peroxidase blocking buffer for 10 min, and then immunostained with antibodies specific for anti-mouse Ly6G (Bio X Cell, BP0075-1) and TNF-α (Santa Cruz, sc-52746) O/N. After washing, Alexa Fluor 488-conjugated anti-mouse IgG (Life Technologies, A-11029) and Alexa Fluor 594-conjugated anti-rat IgG (Life Technologies, A-11007) were used as secondary antibodies and bound for 2 h. To suppress auto-fluorescence, Sudan Black B treatment was performed, and the slides were mounted with DAPI (Sigma-Aldrich, D9542) for nuclear counterstaining. Confocal images of neutrophils and TNF-α visualization were taken with a Leica TCS SP8 confocal system and processed with Leica Application Suite X software. The number of anti-Ly6G-positive neutrophils was counted manually from six random fields of confocal images and the integrated intensity of TNF-α fluorescence from multiple confocal images of each group was measured using Metamorph NX 2.0 software.

Immunofluorescence staining of cells was performed in chambered slides (BD Bioscience, San Jose, CA, USA). Cells were seeded in chambered slides and fixed in ice-cold methanol for 10 min. Slides were then washed in phosphate-buffered saline (PBS) three times, and they were incubated with blocking solution (3% BSA in PBS) for 30 min. For immunofluorescence staining of mouse tissues, formalin-fixed, paraffin-embedded sections were used. Slides were deparaffinized and dehydrated. Mouse monoclonal acetylated α-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA) and non-phospho β-catenin antibody (Cell signaling, Danvers, MA, USA) was used as primary antibodies. Samples were then stained with Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 568-conjugated anti-rabbit IgG antibody (Life technologies, Carlsbad, CA, USA) and mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, CA, USA) with DAPI (4′,6-diamidino-2-phenylindole).