Orca flash4.0 lt cmos camera

Sections were washed in 0.1 M PB, then blocked in 10% normal donkey serum with 0.3% Triton X-100 in 0.1 M PB for 1 h before incubating them with primary antibodies (Table 1) at 4 °C in the same blocking solution overnight. On the second day, sections were washed in 0.1 M PB containing 0.3% Triton X-100 and then incubated with fluorophore-conjugated, species-specific secondary antibodies (Table 1) for 2 h at room temperature in 0.1 M PB containing 0.3% Triton X-100 and 10% normal donkey serum. Before mounting sections were washed for 25 min in 0.1 M PB containing 0.3% Triton X-100. Exceptions from this general protocol were made for staining luciferase, p-aSYN, IbA1 and Olig2, where after primary antibody incubation a biotinylated species-specific secondary antibody was used to further improve signal to noise by conjugation with streptavidin. Images were acquired using an AxioImager M2 microscope (Zeiss) equipped with an ORCA-Flash4.0 LT CMOS camera (Hamamatsu C11440-42 U). For confocal images, a TCS SP8 microscope (Leica) was used. Images were processed with FIJI image software [81 (link)] to enhance signal-to-noise. Image data for 3D reconstructions were obtained with a Zeiss Spinning Disc Microscope (Axio Observer Z1) equipped with an Axiocam MRm (Zeiss) and an Evolve 512 EMCCD Camera (Photometrics) and post-processed with ZEN 2012 software (Zeiss).

Live cell wide-field and TIRF imaging for GFP–synaptopodin motility analysis was performed on a Nikon Eclipse Ti-E controlled by VisiView software (VisitronSystems). The microscope was equipped with 488 nm, 561 nm and 640 nm excitation laser lines via a multi-mode fiber. Oblique and TIRF illuminations were achieved with an ILAS2 TIRF system. The angle of the excitation light was adjusted manually to achieve an optimal signal-to-noise ratio. Samples were imaged with a 100× TIRF objective (Nikon, ApoTIRF 100×/1.49 oil). Emission light was captured through a quad-band filter (Chroma, 405/488/561/640) followed by a filter wheel with filters for GFP (Chroma, 525/50 m), RFP (Chroma, 595/50 m), and Cy5 (Chroma, 700/75 m). Multi-channel images were acquired sequentially with an Orca flash 4.0LT CMOS camera (Hamamatsu).

APs were elicited as described above, and the resulting changes in GCaMP6s^{58 (link)} fluorescence were recorded and analyzed. An Orca Flash 4.0 LT CMOS camera (C11440-42U; Hamamatsu Photonics K.K.) with HOKAWO 3.10 software was used for image acquisition. Exposure time was 75 ms. Image series were streamed. Raw data were exported to MS Excel, and ΔF/F was calculated by [F(firing)-F(rest)]/F(rest)^{47 (link)}. Regions of interest (ROI) were chosen in dendrites and axon.

EYFP-H148Q/I152L was used to monitor changes in intracellular iodide concentrations. The fluorescent intensity of this YFP variant decreases with increasing iodide concentrations. HEK293T cells were cotransfected with EYFP-H148Q/I152L and the indicated plasmid at 36 hours before imaging and were plated onto poly-l-lysine–coated coverslips. Cells were washed with PBS and monitored using an imaging system consisting of a DG-5 wavelength switcher (Sutter Instrument), an ORCA-Flash4.0 LT+ CMOS camera (Hamamatsu), and a Ti2 microscope (Nikon). The imaging buffer contained 140 mM NaCl, 3 mM KCl, 2 mM K₂HPO₄, 1 mM CaCl₂, 1 mM MgSO₄, and 5 mM Hepes (pH of 7.4). The fluorescent intensities were quantified with MetaFluor software (Molecular Devices).

SH-SY5Y cells and hippocampal neurons were used for calcium imaging. SH-SY5Y cells were incubated with 10 μM all-trans retinoic acid for 24 h before experiments. Cells were loaded with 1 μM Fura2-AM dye (Thermo Fisher Scientific) for 30 min at 37°C and washed twice using Tyrode’s solution. ABC294640 (30, 60, or 90 μM) and ionomycin (1 μM) were added at the indicated time. Calcium transients were captured by live-cell imaging using a calcium imaging system consisting of a DG-5 wavelength switcher (Sutter Instrument), an ORCA-Flash4.0 LT+ CMOS camera (Hamamatsu), and a Ti2 microscope (Nikon).