Egf alexa fluor 488

For organ culture studies, biopsy fragments from the duodenum were obtained from patients with CD presenting with villous atrophy (12 patients, mean age of 5 years), controls affected by gastroesophageal reflux (12 subjects, mean age of 6 years) and patients with CD on a GFD (10 subjects, mean age of 12 years). The age range for all patients was 2–16 years. Patients with CD on a GFD had negative serology (anti-tTg antibody titers ranging between 0 and 1.5 U/ml and negative for antiendomisum antibodies (EMA)) and a normal biopsy (Marsh T0). Patients with villous atrophy (Marsh T3c) had positive serology (anti-tTg antibodies >50 U/ml and EMA positive). Anti-tTg antibody titers were measured using Eurospital kit EU-tTG (Supplementary Table 1). Patients had been on a GFD for at least 4 years, the adherence to GFD was complete and patients had remission of the symptoms. None of the CD patients was affected by dermatitis herpetiformis. Biopsy fragments were cultivated as previously described^{10 (link)}. Briefly intestinal samples were cultured for 24 h with medium alone or with 100 µg/ml P31-43 (INBIOS, Naples, Italy), 100 ng/ml EGF (Invitrogen, San Giuliano Milanese, Italy) and EGF-Alexa Fluor-488 (20 ng/ml, Invitrogen, San Giuliano Milanese, Italy). Specimens were harvested, snap-frozen in liquid nitrogen, embedded in OCT and stored at −80 °C until required for further analyses.

Cells were fixed with 3% paraformaldehyde or 100% Methanol for indirect immunofluorescence and then permeabilized in 0.1% Triton X-100. Cells were blocked in FDB buffer (5% FBS, 5% goat serum, 2% BSA in PBS, 1 mM MgCl2 and 1 mM CaCl2, respectively), and primary and secondary antibody incubations performed in FDB for 1 hour at room temperature. Cells were observed and images were taken using a confocal microscope (Nikon A1R and Olympus). Primary and secondary antibodies were used as 1:30 of EEA-1 (#2411; Cell Signaling), 1:50 of LAMP1 (ab25630; Abcam), 1:20 of Rab7 (#2094; Cell Signaling), 1:30 of Rab5 (#2143; Cell Signaling), 1:30 of Rab11 (#5589), 1:250 of GM130 (BD Biosciences) and 1:100 of β-tubulin (T4026; Sigma). 1:100 of γ-tubulin (T3559; Sigma), Alexa Fluor 488 (1:200), Alexa Fluor 555 (1:300) and Alexa Fluor 647 (1:50) were from Molecular Probes. For colocalization of EGF with lysosomes and early endosomes, fluorescence intensity ratios of EEA1 or LAMP1 with EGF-Alexa Fluor 488 (Invitrogen) for different time points were calculated using Fiji and signal intensities plotted against time for NCI-H1299 and PC9 cells.

Chemotaxis and cell tracking slides were from Ibidi (Munich, Germany), and glass-bottom dishes were from MaTek (Ashland, MA). A murine monoclonal antibody to NAV3 was generated in our laboratory. An anti-EGFR for immunoblotting was from Alexis Biochemicals (Lausanne, Switzerland). Secondary antibodies and Alexa Fluor 488-EGF were from Invitrogen (Eugene, OR). The cDNA of human NAV3 was purchased from OriGene (Rockville, MD).