Cfi plan apochromat lambda 20

The fully automated microscope used for the experimental setup was a Ti2 Eclipse (Nikon) with a SOLA SE II (Lumencor) light source and a DIQ2 camera (Nikon) for fluorescence measurements. A temperature box around the microscope provided a stable temperature of 37 °C and an atmosphere chamber provided an atmosphere of 5% CO₂ with a humidity of at least 70%. The objective used for all measurements was a CFI Plan Apochromat Lambda 20× (Nikon). We used filter sets for DAPI and GFP from Nikon. For PE and the two barcode fluorophores, we used custom cubes with filter sets 532/10-552-575/35, 635/10-649-670/30, and 635/10-649-711/25 (emission filter, dichroic mirror, and excitation filter), respectively. The illuminance values for DAPI, GFP, barcode 1, and barcode 2 were set to 2%, 25%, 33%, and 33%, respectively. The exposure times for DAPI, GFP, barcode 1, and barcode 2 were 100 ms, 250 ms, 100 ms, and 100 ms, respectively. The exposure time for PE illumination varied depending on the experiment. For the alamarBlue™ assay, the exposure time was set to 100 ms and the light intensity was set to 15% of the maximum power. For the bead evaluation, the light intensity was set to 50% and the exposure time to 500 ms.

Primary neurons on cover glasses or 20 μm cryostat-sectioned coronal brain slices were incubated for 1 h at room temperature with blocking buffer (PBS containing 5% [v/v] goat serum [catalog no.: 16210-064; Gibco, New Zealand], 1% [w/v] bovine serum albumin, and 0.3% [v/v] Triton X-100), followed by 2 h of incubation with primary antibodies (Table 1). After washing in PBS, the samples were incubated with AlexaFluor-conjugated secondary antibodies, washed again, mounted for microscopy with ProLong Glass Antifade (catalog no.: P36980; Invitrogen), and examined under a Nikon A1RHD confocal microscope with a CFI Apochromat TIRF 100× Oil, numerical aperture 1.49 or CFI Plan Apochromat Lambda 20×, numerical aperture 0.75 (Nikon Instruments, Tokyo, Japan). Images were acquired with NIS-elements (Laboratory Imaging, Nikon) and then processed in ImageJ (NIH, Bethesda, MD).