Alexa fluor 488 anti mouse igg

WT- and 3Tg-iAstro cells, grown on 13 mm glass coverslips, were fixed in 4% formaldehyde, permeabilized (7 min in 0.1% Triton X-100 in phosphate-buffered saline (PBS)) and immunoprobed with an appropriate primary antibody (diluted in PBS supplemented with 1% gelatin) for 1 h at 37 °C. After 3 times washing in PBS, an Alexa-conjugated secondary antibody (1:300 in PBS supplemented with 1% gelatin) was applied for 1 h at room temperature (RT). The following primary antibodies were used: AQP4 (Alomone Labs, Cat. No. 249-323), Aldh1l1 (Abcam, Cat. No. Ab190298), GS (Abcam, Cat. No. Ab73593), GFAP (Chemicon International, Cat. No. CBL411) and GLT-1 (Alomone labs, Cat. No. AGC-022). Secondary antibodies were as follows: Alexa Fluor 488 anti-mouse IgG, Alexa Fluor 555 anti-rabbit IgG (all secondary antibodies were from Molecular Probes, Life Technologies, Monza, Italy). Nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired by Zeiss 710 confocal laser scanning microscope equipped with EC Plan-Neofluar 40×/1.30 Oil DIC M27 objective and Zen software.

Cells were fixed with 4% paraformaldehyde (PFA) in PBS, permeabilized and blocked with 0.1% Triton-X100 plus and 5% normal goat serum in PBS. Then, they were incubated overnight at 4°C with the following primary antibodies: mouse anti-DLX2, 1:500 (Santa Cruz), rabbit anti-GABA, 1:1000 (Sigma), rabbit anti-GAD65/67, 1:250 (Millipore), mouse anti-GFAP, 1:200 (Millipore), mouse anti-MAP2, 1:200 (Chemicon), rabbit anti-MAP2, 1:1000 (Millipore), mouse anti-nestin, 1:200 (Chemicon), mouse anti-NeuN, 1:500 (Millipore), rabbit anti-neurofilament M, 1:200 (Millipore), mouse anti-NKX2.1, 1:1000 (Millipore), rabbit anti-OLIG2, 1:1000 (a gift from Dr. Charles Stiles, Harvard Medical School), mouse anti-PAX6, 1:250 (Millipore), mouse anti-PSD95, 1:500 (Millipore), rabbit anti-S100, 1:250 (Dako), mouse anti-SOX2, 1:500 (Millipore), rabbit anti-synaptophysin, 1:250 (Sigma), rabbit anti-TuJ1, 1:1000 (BioLegend) and mouse anti-vGAT, 1:200 (Synaptic Systems). After washing with PBS, cells were incubated with the secondary antibodies, Alexa Fluor® 555 anti-mouse IgG (Molecular Probes) and Alexa Fluor® 488 anti-mouse IgG (Molecular Probes). Cells were then counter-stained with 4,6-diamidino-2-phenylindole (DAPI) (Santa Cruz). The images were captured using a confocal laser-scanning microscope (LSM700; Zeiss) and digital inverted fluorescence microscope (DM5000B; Leica).

TRAP staining was carried out with TRAP-staining kit (Primary Cell Co.). For immunostaining, cells were fixed with 4% paraformaldehyde. Then, cells were permeabilized and blocked with PBS containing 2% bovine serum albumin (BSA) and 0.01% Tween 20, and incubated with anti-HIV p24 antibody (clone Kal-1; Dakocytomation) and anti-TRAP antibody (Santa Cruz). After being washed, cells were further incubated with Alexa Fluor 488 anti-mouse IgG (Molecular Probes), Alexa Fluor 546 anti-rabbit IgG (Molecular Probes), and Hoechst33258 (Molecular Probes). The images of fluorescence were acquired by fluorescent microscopy using a BZ-8000 (Keyence). For flow cytometry, cells were harvested with 10 mM EDTA/PBS. After preincubation with a human Fc receptor blocking reagent (MBL), cells were stained with the corresponding antibodies. Fluorescence was analyzed with a FACSCalibur (Becton Dickinson). The following antibodies were used for staining: fluorescein isothiocyanate (FITC) -labeled anti CD4, CD184/CXCR4, CD195/CCR5, CD14, and CD71. In addition, the following isotype controls were used as negative controls for staining for each receptor: FITC-labeled mouse IgG1 for CD4, FITC-labeled rat IgG1 for CXCR4, and FITC-labeled rat IgG2a for CCR5. All of the antibodies except for CD14 and CD71 (Miltenyi Biotech) were obtained from MBL.

One day before TNF-α treatment, HeLa cells were seeded onto glass cover slips. Cells were then treated with 30 ng/ml TNF-α for 1 h, followed by fixation with 4% formaldehyde and 4% sucrose for 20 min at 4 °C. After washing twice with phosphate-buffered saline (PBS), the fixed cells were permeabilized with PBS containing 0.2% Triton X-100 at 4 °C for 15 min. Permeabilized cells were washed three times with PBS and then incubated with PBS-BG (PBS containing 0.1% bovine albumin serum and 3% fetal bovine serum) for 1 h at room temperature. Cells were incubated with the indicated primary antibody diluted in PBS-BG overnight at 4 °C. After washing with PBS, cells were incubated with Alexa Fluor 488 anti-mouse IgG, Alexa Fluor 546 anti-rabbit IgG, or Alexa Fluor 488 anti-goat IgG (1 : 1000 dilution; Molecular Probes, Eugene, OR, USA) diluted in PBS-BG for 2 h at room temperature. Finally, cells were mounted in a solution containing DAPI (Vectashield, Vector Laboratories, Inc., Burlingame, CA, USA) and observed with a laser confocal microscope (Carl Zeiss, Thornwood, NY, USA).