Cy3 anti rabbit igg

In brief, cells were fixed in 4% PFA (Sigma-Aldrich) for 15 min, followed by a 10-min permeabilization step (0.1% Triton X-100 in PBS for internal cell markers). The blocking step was performed by incubation in 2% BSA for 30 min. Cells were incubated with primary antibodies at 4°C overnight and further incubated with secondary antibodies for 1 h. For MitoTracker dye staining, cells were cultured in the normal media with 150 nM MitoTracker red (Invitrogen) for 30 min before further fixation and immunostaining. Antibodies against the following proteins were used at the indicated dilutions: CHCHD2 (HPA027407, 1:200; Sigma-Aldrich), SOX1 (4194S, 1:200; Cell Signaling Technology), NESTIN (MAB5326, 1:200; EMD Millipore), TUJ1 (MRB-435P, 1:500; Covance), mtTFA (ab119684, 1:500; Abcam), anti–mouse IgG-FITC (1:800; Sigma-Aldrich), and anti–rabbit IgG-Cy3 (1:1,000; Jackson ImmunoResearch Laboratories, Inc.). Nuclei were labeled with DAPI (Thermo Fisher Scientific). Colocalization coefficient studies were performed using ImageJ software by calculating Manders’ colocalization coefficient, which describes the amount of colocalizing pixels of GFP using pixels generated by RFP.

The following antibodies were used: homemade anti-Myo1b polyclonal antibodies (Almeida et al., 2011 (link)), 1:1,000 for Western blot and 1:50 for immunofluorescence; mouse monoclonal antibodies against Tau-1 (1:100) purchased from EMD Millipore and β-3 tubulin (1:150) purchased from Abcam; and rabbit polyclonal anti-GAPDH (1:20,000 for Western blot) purchased from Sigma-Aldrich. We also used Alexa Fluor 488–, 546–, or 647–coupled secondary antibodies against mouse or rabbit IgG (1:400) and horseradish peroxidase–conjugated secondary antibodies against mouse or rabbit IgG (1:5,000; Invitrogen); and anti–rabbit IgG Cy3 (1:400; Jackson ImmunoResearch Laboratories, Inc.; or 1:500; Molecular Probes). Alexa Fluor 488– or 546–conjugated phalloidin was used to detect F-actin (1:400; Invitrogen).