Anti rabbit igg tritc

Antibodies against ADAM10 (sc- 48400), ADAM17 (sc- 13973), FAS (sc-1023) and PARP (sc-7150) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody against β-actin (AC-15), rabbit polyclonal antibody against α-tubulin, anti-rabbit IgG-FITC (F0382) and anti-rabbit IgG-TRITC were purchased from Sigma (St Louis, MO). Rat IgG2A anti-ADAM10 conjugated with Phycoerythrin (FAB94GP) was obtained from RD Systems (Minneapolis, MN). Peroxidase anti-mouse IgG, peroxidase anti-rabbit IgG, peroxidase anti-goat IgG and anti-goat IgG-FITC (H + L) antiantibodies were obtained from KPL (Gaithersburg, MD). Anti-mouse and anti-rabbit UltraVision Detection Systems was obtained from Thermo Scientific (Fremont, CA). TRIzol-Reagent and SuperScript II Reverse Transcriptase were obtained from Invitrogen (Carlsbad, CA), Taq polymerase Fermentas (Burlington, Canada).

The cells were cultured with 5 μM ATV in 6-well plates for 2 days and then irradiated. At 3 h after 8 Gy of irradiation, the cells were fixed in 3.7% formaldehyde, blocked with 1% BSA/0.2% Triton-X-100 diluted in PBS, and stained with anti-γH2AX antibody (Abcam, Cambridge, UK) followed by a secondary antibody (anti-rabbit IgG-TRITC, Sigma, St. louis, MO, USA). The cells were then washed, loaded into a pre-assembled cytospin cuvette, and centrifuged at. 1000 rpm for 5 min in Shadon Cytospin 3 (Thermo Scientific, Eugene, OR, USA) to attach Raji cells. The cells were cover-slipped with mounting medium containing a 40, 6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen, Carlsbad, CA, USA) solution (Vector Laboratories, Inc., Burlingame, CA, USA). All groups were then assessed for γH2AX by using confocal microscopy (LSM 710, Carl Zeiss, Germany) with filters specific for DAPI (excitation/emission: 359/461 nm) and TRITC (excitation/emission: 555/580 nm).

To evaluate the expression of stem cell and retinal progenitor markers, immunocytochemistry staining was carried out using the primary antibodies: PAX6 (SC-11357, 1/50), RAX (LS-C53650, 1/200), NESTIN (ab-22,035, 1/100), LHX2 (SC-81311, 1/100) and secondary antibodies Anti-Mouse IgG-FITC (Sigma, AP124F) and Anti-Rabbit IgG-TRITC (Sigma, T6778). Briefly, 5 × 10⁴ cells/well coverslips coated with Matrigel were plated in 24-well plates. One day later, samples were fixed with 4% paraformaldehyde for 30 min at room temperature. Subsequently, these cells were permeabilized by 0.4% Triton X-100 for 30 min and were stained with blocking solution-diluted primary antibodies (BSA, 10 mg/ml) and kept at 4 °C overnight. Then, they were treated with secondary antibodies at 37 °C for 1 h. Furthermore, cell nuclei were stained with DAPI (3 ng/ml, Invitrogen). The images were taken by fluorescent microscope (Olympus, Center Valley, PA, USA) equipped with an Olympus DP70 camera.