Alexa fluor 488 conjugated goat anti rabbit antibody

The formalin‐fixed, paraffin‐embedded tissue sections were treated with antigen retrieval and blocked as preciously described (Huang et al, 2014). The slides were incubated with mouse monoclonal anti‐cytokeratin (1:500 dilution, GTX75521, GeneTex, Irvine, CA, USA), mouse monoclonal anti‐human Foxp3 antibody (1:50 dilution, 14‐4777, ebioscience, San Diego, CA, USA), rabbit anti‐GFP antibody (1:100 dilution, ADI‐SAB‐500, Enzo Life Sciences, Farmingdale, NY, USA), or mouse anti‐IL‐17RB antibody (Wu et al, 2015) (1:500 dilution) overnight at 4°C. For immunohistochemistry assay, HRP‐conjugated rabbit/mouse polymer (Dako REAL EnVision, Dako, Glostrup, Denmark) and liquid diaminobenzidine tetrahydrochloride plus substrate (DAB chromogen, Dako, Glostrup, Denmark) were used for visualization. All slides were counterstained with hematoxylin, and the images were taken and analyzed using an Aperio Digital Pathology System (Aperio, Vista, CA, USA). For immunofluorescence assay, Alexa Fluor 488‐conjugated goat anti‐rabbit antibody (1:100 dilution, A11008, Life Technologies, Carlsbad, CA, USA) was used followed by 4′,6‐diamidino‐2‐phenylindole (DAPI) staining. Slides were then mounted with fluorescence mounting medium (Dako, Glostrup, Denmark), and the images were taken by a confocal microscope (Leica TCS SP5 MP).

Cytofluorimetric analysis was performed either by CD56-APC eFluor 780 direct staining or PSA/AlexaFluor 488 indirect staining on U87-MG cells. In brief, U87-MG cells (2 × 10⁵) were seeded into 60 mm diameter plates and maintained overnight. Then, cells were incubated or not in a hypoxic atmosphere for 72 h. Then, cells were collected, washed, and resuspended in PBS, 2% FBS (106 cells/100 µL). Samples were incubated for 30 min at 4 °C with 10 µL of mouse monoclonal anti-CD56-APC eFluor 780 (47-0567-41, eBioscience, dilution 1:10) or rabbit monoclonal anti-PSA antibody (MBS488177, MyBioSource, San Diego, CA, USA—dilution 1:10), washed and resuspended again in 100 µL of PBS, 2% FBS. The samples stained for PSA were incubated for an additional 30 min at 4 °C with AlexaFluor 488-conjugated goat anti-rabbit antibody (dilution 1:200, A11034, Life Technologies. After being additionally washed with ice cold PBS, cells were resuspended in PBS, 2% FBS, and the samples were acquired on a FACs ARIA II instrument using FACs DiVa software (v.6.1.1, both by Becton Dickinson, Milan, Italy). At least 20,000 events were recorded and analyzed using Flowing software (v2.5.1, Turku Centre for Biotechnology, University of Turku, Turku, Finland). Each experiment was performed independently three times.

HIBCPP cell monolayers grown on Transwell inserts were fixed and permeabilized with methanol at minus 20 °C, followed by pre-chilled acetone. After inserts were excised and placed in a new 6-well plate, cells were blocked with blocking buffer (1% bovine serum albumin in PBS^+/+) for 2 h at room temperature. After blocking, cells were incubated overnight in blocking buffer with rabbit anti-ZO-1 antibody (Proteintech, Rosemont, IL, USA) (1:100) in a humidified chamber at 4 °C, followed by a 2-h incubation with Alexa Fluor-488 conjugated goat anti-rabbit antibody (Life Technologies, Carlsbad, CA, USA) (1:500) at room temperature. Cells were treated with 4′,6-diamidino-2-phenylindole (DAPI) (1 µg/mL PBS) for 5 min at room temperature for nuclear staining. Membranes with cells were mounted on glass slides in a ProLong Diamond Antifade Mountant (ThermoFisher Scientific, Waltham, MA, USA) beneath a glass coverslip. Images were obtained using a Zeiss LSM880 inverted confocal microscope with a 20× objective at the Marley Imaging Core within the University of Arizona (Tucson, AZ, USA).