Te2000 u inverted microscope

Immunofluorescence was performed as previously described [24 (link)], with a primary antibody dilution of 1:500 and a secondary antibody dilution of 1:1000. Pictures used to quantify the number of PSD95 puncta, and their size distributions were taken with a NIKON TE2000U inverted microscope using a 60× oil immersion objective. The number of puncta was detected and quantified with Fiji image-processing software by converting the picture to a binary image, setting a threshold for the pixel size, and running the “analyze particle” function. This analysis was performed on a selection consisting of a 100-μm² rectangle located at 0–20, 20–40, and 40–60 μm from the soma. Representative pictures were taken with a Leica SP8 confocal microscope using a 60× immersion objective and five z-stacks (2.16 µm each).

Volvox carteri f. nagariensis (strain EVE) were grown axenically in Standard Volvox Medium (SVM) (Kirk and Kirk, 1983 (link)) with sterile air bubbling, in a growth chamber (Binder, Germany) set to a cycle of 16 hr light (100 μEm⁻²s⁻¹, Fluora, OSRAM) at 28^∘C and 8 hr dark at 26^∘C. Individual biflagellate cells were extracted from Volvox colonies using a cell homogeniser, isolated by centrifugation with Percoll (Fisher, UK), and inserted into a 25 × 25 × 5 mm glass observation chamber filled with fresh SVM. Cells were captured using micropipettes and oriented so that their flagellar beating planes coincided with the focal plane of a Nikon TE2000-U inverted microscope. Motorised micromanipulators (Patchstar, Scientifica, UK) and custom-made stages facilitated accurate rotation and translation of the cells. The flow field characterisation and pairwise synchronization analyses were imaged using a 40× Plan Fluor objective lens (NA 0.6). A higher magnification 63× Zeiss W Plan-Apochromat objective lens (NA 1.0) was used to conduct separate experiments for the waveform analysis. For each experiment, we recorded videos with a high-speed video camera (Fastcam SA3, Photron, USA) at 1000 fps under bright field illumination.

Tissue sections were deparaffinized twice using xylene treatment (10 min each time), and they were re-hydrated by decreasing the alcohol concentration. After washing the tissue sections in distilled water for 1 h, they were stained by hematoxylin solution for 8 min and by eosin for 3 min. After that, the tissue sections were dipped in 0.2% saturated lithium carbonate solution for 30 s. The eosin solution was then used to stain the tissue sections for 1 min after washing the sections in running tap water. Finally, the H&E staining images were photographed with the Nikon TE2000-U inverted microscope (Japan).

The microscope system consisted of a customized Nikon TE-2000 U inverted microscope combined with a Nikon Apo TIRF oil objective (M = 60×, NA = 1.49). Two diode lasers from the Cobolt 06.01 series, with central wavelengths of 488 nm and 638 nm, were guided into the microscope for TIRF illumination of the blue and red channels, respectively. TIRF microscopy has the advantage of reducing the signal from fluorescent molecules in the bulk solution. TIRF is for the current experiments not strictly needed since the fluorescence from vesicles in the bulk solution is very low, thus other microscopy techniques such as confocal microscopy and standard epifluorescence microscopy could also be used in the single-vesicle assay under these conditions. Fluorescence from the illuminated samples was split by a dichroic mirror, (405/488/532/635 BrightLine®, Semrock) and filtered by emission filters (FF01 512/25 and FF01 650/13 from Semrock). The two fluorescence wavelengths were separated by a Hamamatsu W-WIEW GEMINI (A12801-0) and imaged as two sub-images onto a 2048×2048 Hamamatsu ORCA-Flash4.0 V3 digital scientific CMOS camera (C13440) with a pixel size of 6.5 × 6.5 μm. All measurements were conducted using a 2×2 binning and exposure time of 100 ms.

GCs were fixed in PBS with 2% paraformaldehyde, and fixed on the cover glass coated with poly‐L‐lysine at room temperature for 15 min, and then blocked overnight with PBS containing 10% FBS, 1% BSA, 0.05% Triton X‐100 and 2 mM EDTA. Next, GCs were stained with Rabbit anti‐NF‐κB p65 (Cell Signaling Technology, Beverly, MA, USA, 8242S; 1:100) for 2 h. immunoglobulin G (IgG)‐AF594 (Jackson Immune Research) was used to detect the antibody binding. DNA was stained with Draq5 (Cell Signaling Technology). The laser confocal experiment was carried under a Nikon TE2000‐U inverted microscope.

Intracellular Ca²⁺ measurements were performed as described previously [20 (link)] using an epifluorescence detection system (IonOptix Corp, Milton, MA, USA) mounted to a Nikon TE2000U inverted microscope. Isolated cells were loaded with Fluo‐4 AM (10 µmol·L⁻¹) for 15 min and superfused with normal Tyrode's solution (‘NT’) consisting of (in mmol·L⁻¹): 140 NaCl, 4 KCl, 5 HEPES, 1 MgCl₂, 10 glucose, 2 CaCl₂ (pH 7.4 with NaOH). Excitation of Fluo‐4 was at 480 ± 15 nm, emission was collected at 535 ± 20 nm. Myocytes were field‐stimulated at 1 Hz until steady‐state was achieved. For β1‐adrenergic stimulation, ISO (100 nmol·L⁻¹) was added to the superfusion (starting for 5 min before recording first data). Sarcoplasmic reticulum (SR) Ca²⁺ content was estimated by rapid application of caffeine (10 mmol·L⁻¹).