Cfi plan fluor 40 oil

All live-cell imaging experiments were performed on a Nikon A1 laser-scanning confocal microscope system outfitted with a Tokai HIT stagetop incubator utilizing 40× and/or 60× oil immersion objectives (CFI Plan Apo Lambda 60× Oil, Nikon; CFI Plan Fluor 40× Oil, Nikon). Following transfections and/or treatments, medium was changed to phenol red-free growth medium (Gibco) and cells were allowed to equilibrate on the preheated (37°C and 5% CO₂) stagetop incubator for 10 min prior to imaging. Acute blue light stimulation was achieved by utilizing the 488nm laser line and the stimulation module within Nikon Elements imaging software. Activation duration varied from 1-8 sec and laser power ranged from 1-20% as indicated in different experiments. Stimulation regions of interest (ROIs) were drawn over fields of view prior to image acquisition. Following 2-5 baseline images, laser stimulation was performed and cells were imaged for up to 1 hr post-activation. Timing and order of image acquisition was alternated across experiments between experimental groups. Data presented are representative of at least two independent experiments utilizing three or more biological replicates per experiment.

The mounted slices were imaged under a confocal microscope (A1Rsi; Nikon) using a 20x (CFI Plan Apo Lambda 20×, NA 0.75; Nikon) or 40x objective (CFI Plan Fluor 40× Oil, NA 1.3; Nikon). The fluorescence signals of GCaMP6f, Alexa fluor dye 568, and 647 were sequentially collected, except the co-staining of reelin and Wfs1. Since the fluorescence signal of Wfs1 was much stronger than that of reelin, the signal of Wfs1 could contaminate the signal of reelin. We therefore performed a spectrum excitation over the range of 547–733 nm and isolated the emission signals from two major peaks, which corresponded to reelin and Wfs1, using the “blind unmixing” function in the NIS elements confocal software (Nikon).