Cy2 affinipure donkey anti rabbit igg

Therapy-induced changes in the intracellular localization of ENT1 were assessed using immunofluorescence. Cells were fixed with 4% paraformaldehyde at room temperature for 10 min. Cells were washed with PBS, permeabilized and blocked with 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween solution for 1 h. Cells were then incubated with a primary antibodies directed to human ENT1 (Abcam, Cambridge, MA; 1:100) overnight at 4°C. After incubation with the primary antibody, cells were washed with PBS and incubated with Cy™2 AffiniPure Donkey Anti-Rabbit IgG (Jackson ImmunoResearch Inc., West Grove, PA) with at room temperature for 1 h. DAPI (Thermo Fisher Scientific Inc., Grand Island, NY) was used to stain the cell nuclei at a concentration of 0.5 μg/ml. For negative staining control, primary antibody was omitted during the procedure. Digital images were acquired using a Nikon E600 microscope. The results of the membrane positive immunofluorescence of ENT1 were semiquantitatively assessed and scored as previously described [22 (link)]. Scoring criteria were defined as follows: 0 for negative (0% to 5% staining); 1 for weakly positive (5% to 20% staining); 2 for moderately positive (20% to 50% staining); 3 and 4 for strongly positive (50% to 75% and ≥ 75%, respectively).

ipMGs were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 in PBS and blocked with CAS-Block (Life Technologies, 008130) for 10 min at room temperature and incubated with primary antibody (TMEM119 polyclonal antibody, Invitrogen, PA5-62505; 1:100) in CAS-Block overnight. Cells were washed three times with PBS for 5 min and incubated with secondary antibody (Cy2 AffiniPure donkey anti-rabbit IgG, Jackson Immunoresearch, 711-226-152; 1:200) in CAS-Block for 1 h at room temperature. After washing three times with PBS for 5 min each, cells were stained with DAPI, washed once with PBS, overlaid with PBS and imaged with an Olympus IX83-based Live-Imaging system equipped with a VisiScope CSU-W1 spinning disk.