Cyanine 3 conjugated mouse anti gfap igg antibody

NSPs and differentiating cells were cultivated on poly-L-ornithine-coated coverslips and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), followed by a permeabilization with 0.05% Triton X-100 in PBS. We performed a blocking step in PBS containing 3% bovine serum albumin (BSA) at 20–24 °C for 1 h. We conducted immunostainings by incubating cells with primary antibodies diluted in blocking solution overnight at 4 °C, followed by incubation with the corresponding secondary antibodies diluted in the blocking solution for 1 h at room temperature (1:1000; Jackson ImmunoResearch, West Grove, PA, USA). We used the following antibodies: rabbit anti-CD44 IgG (1:100; Abcam, Waltham, MA, USA), rabbit anti-Kir4.1 IgG (1:100; Alomone Labs, Jerusalem, Israel), rabbit anti-GLT1 (SLC1A2) IgG (1:100; Abcam), rabbit anti-GLAST (SLC1A3) IgG (1:100; Abcam), mouse anti-MAP2 IgG (1:200; Chemicon, Rolling Meadows, IL, USA) and mouse anti-O4 IgM (1:100; Bio-Techne). For GFAP staining, we used a cyanine 3-conjugated mouse anti-GFAP IgG antibody (1:400; Sigma, St. Louis, MO, USA). Cells were then washed and mounted with DAPI-Fluoromount G (SouthernBiotech, Birmingham, AL, USA). Images were collected by confocal microscopy using a LSM 510 confocal microscope (Carl Zeiss Micro Imaging, Jena, Germany) and analyzed on Adobe Photoshop (San Jose, CA, USA) software.

NSPs were cultured on poly-L-ornithine coated coverslips and then fixed with paraformaldehyde (4% in phosphate buffer saline (PBS)), followed by a permeabilization with 0.3% Triton X-100 in PBS. After washing in PBS, the blocking step was done in PBS containing 3% BSA at room temperature for 30 min. The cells were then incubated overnight at 4 °C with primary antibodies, purified mouse anti-human KI67 (1:100; BD Biosciences, Allschwil, Switzerland #550609), rabbit anti-CD133 IgG (1:100; Abcam, Cambridge, UK #ab19898), rabbit anti-CD44 IgG (1:100; Abcam, Cambridge, UK #ab243894), and rabbit anti- NF-κB p65 (1:100; cell signaling technology, Danvers, MA, USA #8242). After washing, cells were incubated with the corresponding anti-rabbit or anti-mouse secondary antibodies conjugated to cyanine 2 or cyanine 3 for 1 h at room temperature (1:1000; Jackson ImmunoResearch, Cambridge, UK). For the GFAP staining, a cyanine 3-conjugated mouse anti-GFAP IgG antibody (1:800; Sigma C9205) was used. Cells were then washed and mounted with DAPI-Fluoromount G (SouthernBiotech, Birmingham, AL, USA). The immunoreactivity was assessed using a LSM 510 META inverted confocal microscope (Carl Zeiss Micro Imaging, Oberkochen, Germany) and analyzed on ImageJ software.