Fluoview fv1200 microscope

The TUNEL assay was carried out using Click-iT TUNEL Alexa Fluor 647 kit (Thermo Fisher Scientific, Middletown, VA, USA), according to the manufacturer’s instructions. Briefly, HeLa cells at a concentration of 1.5 × 10⁵ cells/mL were cultured in glass-bottom confocal culture dishes, and incubated under 5% CO₂ for 24 h at 37 °C. The cells were rinsed with PBS and treated with (0, 1.0, and 10) µM of β-cryptoxanthin for 24 h, and then incubated with 4% (v/v) formaldehyde for 20 min, followed by 0.25% (v/v) Triton X-100 for 25 min. TUNEL images were obtained by Olympus FLUOVIEW FV1200 microscope (Olympus Corporation, Japan). The TUNEL-positive cells were assessed by manual counting of at least 300 cells. Cells with only nuclear staining were considered TUNEL-positive (cytoplasmic staining was not considered).

Animals were anaesthetised with ketamine/xylazine and transcardially perfused with PBS, followed by 4% paraformaldehyde. Brains were removed from the skull and post-fixed overnight. Free-floating 40-μm sections were cut on a cryostat and processed as previously described.^{19 (link)} The following antibodies were used: primary- rat anti-BrdU (1:200; Axyl, Westbury, NY, USA), mouse anti-NeuN (1:1000, Millipore, Ontario, Canada), secondary-goat anti-rat Alexa Fluor 488 (1:200; Life Technology, Thermo Fisher Scientific, Ontario, Canada) and goat anti-mouse Rhodamine Red X (1:200; Jackson Lab, West Grove, PA, USA).
Cell counting was performed in the dentate gyrus granule cell layer and the 50-μm border along the hilar margin. Stained BrdU+ nuclei were scored in every sixth section throughout the rostrocaudal extent of the granule cell layer. All BrdU+ cells were counted bilaterally in each of the sections using a Nikon Eclipse E600 microscope and the average values for each animal were considered. For confocal microscopy, an Olympus Fluoview FV1200 microscope was used. In this study, we did not investigate subventricular neurogenesis, as this has not been affected by DBS in previous work.^{21 (link)}Location of electrode tracks was confirmed with cresyl violet staining. Animals with misplaced electrodes or lost caps were excluded from the study.

Retinal samples were obtained from the two groups of mice with different adaptation patterns, consistent with the conditions of light/dark-adapted single-cell sequencing samples. Eyeballs were collected at 2:00 pm (ZT6), placed in 4% paraformaldehyde (PFA) solution, fixed at room temperature for 2 h, and transferred to 30% sucrose in PBS overnight at 4°C. The cornea and lens were dissected and removed using a frozen microtome (Lecia) to obtain 16-μm retinal sections. The sections were postfixed with 4% PFA and then assayed using an RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD) according to the manufacturer’s instructions. A Fluoview FV1200 microscope (Olympus) was used for image recording, and ImageJ was used for image analysis.

Virus was incubated with 0.65 M CaCl₂ and 1 nM biotinylated Cy3B-labelled DNA before being immobilized via neutravidin on the surface of a pegylated glass slide. Viruses were fixed in 4% paraformaldehyde and permeabilised with 0.5% Triton-X-100, before being incubated with primary and secondary antibodies. A/Udorn/72 virus was stained with an anti-NA primary antibody and Alexa647-labelled secondary antibody and A/WSN/33 virus was stained with an anti-NP primary antibody and Alexa647-labelled secondary antibody. Localisations in each channel were identified using the NanoImager software suite.
MDCK cells were grown on 13 mm coverslips in 24-well plates before being infected with A/Puerto Rico/8/1934 virus at a multiplicity of infection of ~20 × 10⁶ PFU/mL. The virus was incubated with 0.65 M CaCl₂ and 1 nM Atto647N-labelled DNA before being added to the cells. The cells were incubated for 1 hour at 37 °C to allow viruses to adhere before being fixed for 15 minutes in 4% paraformaldehyde and mounted onto glass microscope slides with Mowiol containing DAPI. Cells were viewed using an Olympus Fluoview FV1200 microscope and images processed with ImageJ.

ROS production induced by different nano particles in KYSE-450 cells induced was evaluated with DCFH-DA cell-permeative probe. DCFH-DA (Dichloro-dihydro-fluorescein diacetate) is cleaved by cellular esterase and oxidized by ROS to yield green fluorescence in the cytoplasm. Briefly, ESCC KYSE-450 cells were seeded on Millicell EZ slides, and stained with 25 µM of DCFDA purchased from Sigma (D6883) for 40 min. The cells were washed with PBS for 3 times, and cultured back in RPMI-1640 full growth media and treated with different nano particles for 12 h. Cell nuclei were stained DAPI (1 µg/µl) for 10 min, then washed with PBS before fluorescence microscope observation with excitation/emission at 495 nm/529 nm and 358 nm/461 nm respectively. Fluorescent images were taken with Olympus Fluoview FV1200 microscope at the magnification of 40×.

TE-13 cells were incubated with 2-AAPA at 37°C in a humidified atmosphere of 5% CO₂ for 4 h. The immunofluorescence staining of microtubules were processed as described earlier [25 (link)]. Briefly, the TE-13 cells were fixed in 4% paraformaldehyde at room temperature for 1 h. The cells were washed 3 times with PBS and incubated with cell permeable solution (0.1% Na-citrate, 0.1% Triton-X-100 in PBS) at room temperature for 1 h. After incubation with the blocking solution (5% bovine serum albumin in PBS) overnight at 4°C, microtubules were visualized by incubating with a mouse monoclonal anti-α-tubulin-FITC (1:200) at 37°C for 2 h, and nuclei were stained with DAPI (1 μg/mL). Fluorescent images were taken with an Olympus Fluoview FV1200 microscope. Paclitaxel and vincristine sulfate were employed as positive controls for microtubule stabilization and depolymerization, respectively.