Af546

Blood was collected from retro-orbital puncture into 40 µM D-phenylalanyl-prolyl-arginyl chloromethyl ketone, 5 U/ml heparin, and 40 U/ml fragmin. Samples of 400 µl were preincubated with DiOC₆ (0.5 µg/ml, Anaspec, Fremont, CA, USA) and fibrinogen AF546 (25 µg/ml, Thermo-Fischer Scientific, Waltham, MA, USA) for 5 min before whole blood perfusion. Coverslips coated with micro-spots of type I collagen (1 µl, 100 µg/ml, Nycomed, Hoofddorp, The Netherlands) were mounted on a transparent, parallel plate flow chamber (50 µm depth, 3 mm width, and 300 mm length). Flow perfusion using a shear rate of 1000 s⁻¹ was performed on all samples^{67 (link)}. Brightfield and fluorescence images were captured every 30 s for 3.5 min using an EVOS microscope, equipped with a 60× objective. Surface area coverage was analyzed using semi-automated scripts operating in Fiji (ImageJ)^{68 (link)}.

Monobiotinylated Fab′ fragment of anti-mouse IgM+G Ab (mB-Fab′–anti-Ig) was made from the F(ab′)₂ (Jackson ImmunoResearch Laboratories) as described before [39 (link)]. The disulfide bond that connects the 2 Fab′ was reduced using 20 mM 2-mercaptoethylamine and then biotinylated by maleimide-activated biotin (Thermo Scientific). Fab′ was purified by using Amicon Ultracentrifugal filters (Millipore) and examined by a biotin quantification kit (Thermo Scientific) and then conjugated with AF546 (Invitrogen). To stimulate B cells with sAg, B cells were incubated with AF546–mB-Fab′–anti-Ig (2 μg/ml) together with mB-Fab′–anti-Ig (8 μg/ml) for 30 min and streptavidin (1 μg/ml) for 10 min at 4°C. Streptavidin was omitted as a negative control. The cells were washed and warmed up to 37°C for different time points. To stimulate B cells with mAg, cells were incubated with AF546–mB-Fab′–anti-Ig and mB-Fab′–anti-Ig tethered to lipid bilayers with streptavidin at 37°C for different time points. As a control, B cells were incubated with AF546–Fab–anti-mouse IgM+G (2 μg/ml) at 4°C and then incubated with transferrin (Tf)-tethered lipid bilayers, on which the density of Tf was equal to that of AF546–mB-Fab′–anti-Ig.

Cells were cultured on glass coverslips and were allowed to attach for 24 hours prior to the initiation of treatment. Cells were fixed with 4% paraformaldehyde following permeabilization with 0.02% TritonX-100. Cells were blocked with 5% goat serum (GS). Incubation with primary antibodies against HIF-1α (Abcam) and Transferrin Receptor (Invitrogen) was performed at 1:100 dilution in 5% GS for 1 hour. Cells were washed with phosphate buffer saline (PBS) and incubated for 30 minutes with the secondary fluorescent antibodies (AF488 and AF546, Invitrogen; 1:500 in 5% GS), then incubated with DAPI (1 μg/ml; SIGMA) for 5 minutes. Cells were washed with PBS and mounted onto microscope slides in mounting media. Slides were imaged in Zeiss LSM 800 Airyscan Confocal microscope. Co-localization was analyzed using confocal microscopy determining a line profile of fluorescence intensity. The average fluorescence intensity of several line profiles was obtained to analyze differences in HIF-1α nuclear localization in control versus CCA-treated cells. All steps were performed at room temperature.

Tooth pulp cryosections were stained with Fibronectin and FnBPA5 probe as previously described.^{39 (link)} Briefly, thawed sections were washed with ice-cold 1xDPBS and blocked with 0.3 mol·L^-1 glycine animal-free blocking buffer (Animal-Free Blocker® and Diluent, R.T.U. SP-5035-100) at room temperature for 30 min. Sections were then incubated with 1 μg·mL^-1 Cy5-FnBPA5 or Cy5-labelled scrambled-FnBPA5 (scrambled-FnBPA5) for 1 h, followed by three 5 min washes with Dulbecco’s phosphate-buffered saline (DPBS), second blocking with the blocking buffer and incubation with polyclonal rabbit anti-Fibronectin (ab23750, Abcam) and monoclonal rat anti-Collagen I (7025, Chondrex) antibodies, overnight at 4 °C. The following day samples were extensively washed with DPBS before 1 h incubation with secondary antibodies and 2 μg/ml DAPI at room temperature. The secondary antibodies used were goat anti-rabbit (AF488, A11034, Invitrogen) and goat anti-rat (AF546, A11081, Invitrogen) antibodies. Following extensive washes after incubation with the secondary antibody, the samples were mounted using DAKO Fluorescence mounting medium (DAKO, Denmark) and imaged.