Fitc conjugated secondary antibody

Cells derived from mouse tumors were fixed with 4% paraformaldehyde for 10 min, then stained with primary antibodies against SYP (Abcam, #32127) at 1:600 dilution, CgA (Invitrogen, Waltham, MA, USA, #MA5-13096) at 1:400 dilution, and SSTR2 (Sigma, St. Louis, MO, USA, #HPA007264) at 1:400 dilution overnight. Cells were washed and incubated with an FITC-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, #115-095-062 and #711-095-152) at 1:500 dilution for 1 h. Immunofluorescent images were taken using a fluorescent microscope at 200 ms exposure time.

For BrdU DNA replication analysis PC Cl3 cell cultures grown on coverslips were supplemented with 10 mM bromodeoxyuridine (BrdU; Roche, Basel, Switzerland) for 15 min. Subsequently, cells were fixed in 3 % paraformaldehyde (Sigma) and permeabilized with 0.2 % Triton X-100 (Sigma). The coverslips were incubated with anti-BrdU antibody followed by FITC–conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). Nuclei were counterstained with HOECST (Sigma). For growth curve analysis PC Cl3 cells were grown in 6-well dishes. The number of cells was counted in triplicate from control and SOD3 wells in each analysis date by burker chamber using formula: number of cells/ml = average of cells in each square × 10⁴ × dilution factor.

Cell proliferation was measured by the incorporation of the thymidine analogue 5′-bromo-2-deoxyuridine (BrdU) that is incorporated into the DNA of dividing cells in immunohistochemically detectable quantities during the S phase of cell division. After MCAO, the animals were twice injected intraperitoneally with BrdU (Roche) (50 mg/kg at a concentration of 10 mg/mL in 0.9% NaCl) at 24 hours and 1 hour before sacrifice. For immunohistochemical detection of incorporated BrdU, double-stranded DNA was denatured to a single-stranded form suitable for immunohistochemical detection on sections. Sections were incubated in 50% formamide in standard sodium citrate at 65°C for 2 hours and treated further with 2 M HCl at 35°C for 30 min. After being rinsed for 10 min at room temperature in 0.1 M boric acid, the sections were washed with PBS and then incubated with 0.03% H₂O₂ in methanol for 5 min. The sections were incubated with a primary antibody against BrdU (1 : 1,000, Roche Diagnostics) at room temperature for 30 minutes, washed with PBS, and reacted with a FITC-conjugated secondary antibody (1 : 200, Jackson ImmunoResearch, PA, USA) for 30 min at room temperature. After washing, stained tissue samples were mounted using Vectashield mounting medium (Vector Lab, Burlingame, CA, USA).

For antigen retrieval, the spheroid and tissue sections were reacted with citrate solution for 20 min (10 mM, pH 6.0). Following nonspecific region blockade with normal goat serum (Sigma) and triton X, the sections were reacted with primary antibodies against E-cadherin, PCNA, CD31, and α-SMA (these antibodies were purchased from Abcam) overnight at 4 ℃. Then the samples were washed using PBS, followed by incubation with FITC-conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA) for 1 h at room temperature. For nucleus staining, DAPI was used for the samples. Lastly, the samples were photographed using a fluorescent microscope (Leika).

To analyse DNA replication, 10 mM bromodeoxyuridine (BrdU) (Roche, Basel, Switzerland) was added to the growth medium for a 15-minutes treatment. Subsequently, the cells were washed three times with PBS, fixed in 3% paraformaldehyde (Sigma, St. Louis, MO, USA) for 20 min at −20 °C, washed three times with PBS, and stained using anti-BrdU (Roche) primary antibody, FITC–conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), and Hoechst nuclear stain (Sigma). BrdU positive cells were counted from 30 microscope high power fields.