Rhodamine conjugated anti mouse igg cy3 antibody

Cells were grown at density of 6 × 10⁴ on glass coverslips 12 mm diameter round. Cells were fixed at different time points after differentiation (3, 7 and 10 days) with 4% formaldehyde solution for 15 min at room temperature. Cells were then washed twice with PBS 1 ×, pre-incubated in blocking solution (BSA 5 mg/mL and Triton 0.1% in PBS 1 ×) for 15 min, and incubated overnight with anti-Microtubule-associated protein 2 (MAP-2) monoclonal antibody 1:400 (M4403, Sigma-Aldrich) diluted in blocking solution. The day after, cells were washed twice with PBS 1 ×, and incubated for 1 h with a rhodamine-conjugated anti-mouse IgG Cy3 antibody 1:150 (115–165-003, Jackson ImmunoResearch). After two washing with PBS 1 ×, the coverslips were mounted on slides and examined, using the 20 × objective, under a fluorescence microscope (DMRBE, Leica Microsystems), equipped with digital video camera (Spot-RT Slider, Diagnostic Instruments, Mi, USA). The images, acquired in Tiff format, were adjusted for brightness and contrast with the camera software (SPOT Advanced software, v. 4.0.9, Diagnostic Instruments). The specificity of the primary antibody was assessed by analyzing the differential basal expression of MAP-2 protein in undifferentiated and differentiated SH-SY5Y cells (low and high, respectively), and in negative Hep G2 cells (data not shown).

Cells were grown at a density of 6 × 10⁴ on glass coverslips 12 mm diameter round. Cells were fixed in their undifferentiated state or 11 days after differentiation with 4% formaldehyde solution for 15 min at room temperature. After two washes with PBS 1×, cells were pre-incubated in blocking solution (BSA 5 mg/mL and Triton 0.1% in PBS 1×) for 15 min and incubated overnight with anti-Microtubule-associated protein 2 (MAP-2) monoclonal antibody 1:400 (M4403, Sigma-Aldrich, St. Louis, MO, USA) diluted in blocking solution. After two washing with PBS 1x, fluorescent signal was visualized following 1 h incubation with a rhodamine-conjugated anti-mouse IgG Cy3 antibody 1:150 (115–165–003, Jackson ImmunoResearch). After two washes with PBS 1×, the coverslips were mounted on slides and examined, using the 20 × objective, under a fluorescence microscope (DMRBE, Leica Microsystems) equipped with digital video camera (Spot-RT Slider, Diagnostic Instruments, Sterling Heights, MI, USA). The images were adjusted for brightness and contrast with the camera software (SPOT Advanced software, v. 4.0.9, Diagnostic Instruments, Sterling Heights, MI, USA).