Te2000 u fluorescence microscope

MDA-MB-231 and MRC-5 cells were infected with SG400-EGFP at a multiplicity of infection (MOI) of 1, and were observed under the TE2000-U fluorescence microscope (Nikon) [15 (link)]. Photographs were taken at 24, 48, and 72 h after infection.

For fluorescence microscopy, NO and H₂O₂ were visualized using Diaminofluorescein-FM diacetate (DAF-FM-DA) and 2′,7′-dichlorofluorescein diacetate (H₂DCF-DA) probes, respectively (Sigma-Aldrich). Briefly, leaf dices were first loaded with 15 μM DAF-FM-DA for 30 min or 50 μM H₂DCF-DA for 10 min in Tris/KCl loading buffer (pH 7.2). The process was performed in darkness at 25 °C. Further, leaves were washed three times (5 min each) using Tris/KCl loading buffer (10 mM Tris and 50 mM KCl, pH 7.2), and visualized under a TE2000-U fluorescence microscope (490 nm excitation; 515 nm emission) (Nikon, Tokyo, Japan). Treatments were repeated at least five times. NO and H₂O₂ contents were also detected using a Griess reagent (Sigma-Aldrich) and an Amplex red hydrogen peroxide/peroxidase assay kit (Invitrogen), respectively [33 (link)].

Tissues were fixed in 4% buffered paraformaldehyde, after which they were frozen and then cryosectioned for immunofluorescence staining. Sections of 40 μm depth were stained with a mouse monoclonal anti-HSP72 (i.e., the inducible form of HSP70, at 1:100 dilution, ADI-SPA-810, Stressgen, Enzo, NY, USA). Briefly, sections were hydrated in PBS and cells were permeabilized with 0.5% Tween-20. Blocking was done using 3% BSA for 30 min. Sections were then incubated in the anti-HSP72 antibody at 4 °C overnight. After washing with PBS, sections were then incubated in the appropriate fluorochrome-conjugated secondary antibody (at a 1:400 dilution) for one hour. They were then mounted with Fluoro-Gel II containing DAPI. Images were acquired using a Carl Zeiss LSM700 confocal scanning microscope (Jena, Germany). To quantify the percent of HSP70-positive cells, images were taken using the Nikon TE2000-U fluorescence microscope at 20X magnification. Images were acquired at 10 different locations for each sample, after which the number of HSP70-positive cells and the total number of cells (identified via the DAPI staining) were then counted. Data was presented as % HSP70-positive cells.

Cells were cultured on cover slips with Ang II and with or without PPD for 24 h. Cells were then washed with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton-X for 15 min, and blocked with 5% normal goat serum at room temperature for 1 h. The cover slips were incubated with rabbit polyclonal SIRT1 antibody overnight at 4°C. The cells were washed with PBS and incubated with a fluorescence secondary antibody at room temperature for 1 h. After washing with PBS, the cells were stained with DAPI for 5 min and reviewed, and images were acquired with a Nikon TE-2000U fluorescence microscope (magnification, × 400).

For spontaneous differentiation, the iPSC colonies were fragmented and transferred to a petri dish (SPL Lifesciences, Pochon, Korea) containing an embryoid body (EB) culture medium (DMEM/F12, 10% KnockOut™ Serum Replacement, 1% NEAA, 1X P/S and 0.1 mM β-mercaptoethanol (all from Invitrogen)). The resulting EBs were cultured for 5-10 days in suspension and were transferred onto matrigel-coated slides for 15 days of adherent culture in differentiation medium (DMEM/F12, 1% NEAA, 1X P/S, 0.1 mM β-mercaptoethanol, and 10% FBS (all from Invitrogen)). The cells were immunostained with representative markers of the three germ lineages and were observed under a TE2000U fluorescence microscope (Nikon Corporation, Tokyo, Japan).

These metal-surface adhering bacterial biofilms were also imaged using confocal laser scanning microscopy (CLSM) but clearer images were collected using transparent microscope coverslips. Biofilm-bound and stained glass-slide substrates were incubated for 10 min in the dark and fixated using diluted glycerol/PBS solution before imaging with the aid of a Nikon TE2000-U fluorescence microscope. The P. gingivalis biofilm stained were exposed to the Filmtracer™ LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific).^{45 (link)} This kit was used based on the efficiency of its two-color fluorescence assay for determining bacterial viability. For visual data collection, the bacterial cells were close enough to the surface, so there was no need to view stack images within the biofilm depth using the ZEN lite Digital Imaging Software.

Mice liver sections were deparaffinized, rehydrated, and incubated with 3% H₂O₂ for 10 min. After blocking with 1% BSA for 1 h, the samples were incubated with anti-ferritin (abcam, ab287968, Cambridge, UK) or anti-transferrin (abcam, ab214039, Cambridge, UK) antibody overnight at 4 °C and then incubated with a fluorescein isothiocyanate-conjugated secondary antibody for 2 h. Immunoreactions were visualized under a TE2000-U fluorescence microscope (Nikon, Melville, NY, USA).