Micro max 1300yhs camera

Cells were washed with PBS, fixed with 4% PFA, treated with glycine 0.2 M, permeabilized with Triton X-100 at 0.2% and then blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich) and 5% goat serum. Slides were mounted on Fluoromount-G (SouthernBiotech, 0100-01) and stained with 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; ThermoFisher Scientific, D1306). Immunolabeling was performed at 4°C overnight with the following primary antibodies: rabbit anti-MAP2 (1/25, Cell Signaling Technology, 4542), rabbit anti-Nestin (1/200, Millipore, AB5922), mouse anti-Neurofilament-M and Neurofilament-H (1/50, Millipore, MAB1592), mouse anti-CD14 (1/5, Immunotech, IOM2) and mouse anti-Tubulin (1/100, Zymed Laboratories, Invitrogen, 13-8000). Immunodetection was performed using species and subclass specific Alexa Fluor-405, Alexa Fluor-488 or Alexa Fluor-647 conjugated secondary antibodies (1/200, Life Technology). Antibodies were diluted in PBS containing 0.1% saponin and 0.3% BSA. Actin was stained with rhodamine phalloidin (1/200, Molecular Probes, Invitrogen, R415). Images were visualized under a wide-field microscope (Leica DMI 6000) equipped with a Micro MAX-1300YHS camera using an HCX PL APO 60X oil objective (Princeton Instruments). Images were acquired using Metamorph Software (Version 7.1.3; Molecular Devices).

After cell fixation at DIV 14 (4% paraformaldehyde in PBS, 7 min), cells were permeabilized (0.1% Triton X-100/PBS, 3 × 15 min, PBST) and unspecific binding was blocked (5% normal goat serum (NGS) /PBST, 1 h). Primary antibody incubation was performed in 5% NGS/PBST overnight at 4 °C. After washing with PBST (3 × 5 min), secondary antibody incubation was performed with Alexa Fluor-conjugated antibodies in PBST, 1 h at room temperature. The cells were washed 2 × 10 min with PBST and 1 × 10 min PBS and mounted in Mowiol (SIGMA-ALDRICH). Cells were visualized with a Olympus IX81 epifluorescent microscope, MicroMax:1300YHS camera (Princeton Instruments) and MetaMorph software (Molecular Devices). The exposure time for the BiFC of Venus was 0.5–1 s. Image panels were analyzed with the Fiji Software and prepared using Adobe Photoshop software.