Fitc conjugated goat anti mouse igg

HeLa cells were cultured in DMEM (Dulbecco’s modified Eagle medium, Invitrogen, USA) supplemented with 10% FBS (fetal bovine serum, Invitrogen, New Zealand), 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C with 5% CO₂. Some of these cells were transfected with p3XFlag-CMV14 empty plasmid vector (Flag-HeLa) and p3XFlag-INMAP-CMV14 recombinant vector (Flag-INMAP) [10 (link)], respectively. Flag-HeLa and Flag-INMAP cells were cultured in DMEM with 600 ng/μL geneticin G418 (Merck, USA). The expression of INMAP was detected in stable single cell clones using a Flag monoclonal antibody and an INMAP polyclonal antibody.
Several mouse monoclonal antibodies, including anti-Flag (MBL, Japan) anti-His (MBL, Japan) and anti-GAPDH (MBL, Japan) antibodies, rabbit monoclonal antibodies including anti-p21 (CST, USA), anti-p53 (CST, USA), anti-γH2AX (Bioworld, USA), anti-Bcl-2 (Santa Cruz, USA) antibodies and mouse polyclonal anti-INMAP (Beijing Normal University, China) antibody were used in immunoblot, immunoprecipitation and immune fluorescence experiments. Mouse monoclonal anti-PCNA antibody was provided by Dr. Jian Kuang (University of Texas M. D. Anderson Cancer Center, USA). TRITC-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG were obtained from Vector Laboratories (Peterborough, UK).

Paraffin-embedded tissue sections were deparaffinized and hydrated through a series of graded ethanol solutions, followed by 0.1 M Tris, pH 7.6. Slices were blocked for 1 h at room temperature with 3 % normal goat serum, incubated with primary antibodies including mouse anti-GFAP (1:500, Millipore) and rabbit anti-LRP1 (1:200; Santa Cruz Biotechnology) overnight at 4 °C. After extensive rinsing, sections were incubated for 2 h at room temperature in a mixture of FITC-conjugated goat anti-mouse IgG (1:150, Vector laboratories) and Texas Red-conjugated goat anti-rabbit IgG (1:150, Vector Laboratories). The sections were washed for 3 × 5 min in PBS containing 1.5 μM 4',6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA) and then cover-slipped with buffered PBS/glycerol.

For immunofluorescent assay, the cultured astrocytes were fixed with 4% paraformaldehyde in 10 mM phosphate-buffered saline (PBS; pH 7.4) for 20 min at room temperature, blocked with 3% normal goat serum (Sigma-Aldrich) for 30 min at room temperature, and incubated with rabbit anti-GR antibody (1:100, monoclonal, Cell Signaling, Boston, MA, USA) and mouse anti-GFAP antibody (1:200, monoclonal, Abcam, Cambridge, UK) overnight at 4°C. Then, the cells were washed with PBS, incubated with TRITC-conjugated goat anti-rabbit IgG (1:200; Vector, Burlingame, CA, USA) and FITC-conjugated goat anti-mouse IgG (1:200; Vector) secondary antibodies for 60 min while shaking at room temperature in darkness, and washed with PBS. Finally, the astrocytes were incubated for 2 min with nucleic acid stain, Hoechst33258 (Invitrogen, Carlsbad, CA, USA), diluted 1:5000 in PBS. All primary and secondary antibodies were diluted in PBS containing 1% normal goat serum. The images were captured using a confocal laser scanning microscopy (LSM 710, Carl Zeiss, Germany).