80i fluorescence microscope

Cells were seeded on coverslip coated with 1% gelatin, and then fixed in 4% of formaldehyde for 15 min at room temperature (RT). After washing with PBS, fixed cells were permeabilized with 0.1% Triton X-100 in PBS (PBST) for 10 min and blocked for 1 hr with 5% normal goat serum (NGS) in PBST at RT. Cells were incubated with primary antibodies diluted in 1% NGS in PBST overnight at 4°C. Cells were then washed with PBST and incubated with fluorophore-labeled secondary antibodies for 1 hr at RT. DNA were stained with DAPI. Images were captured by Nikon 80i Fluorescence Microscope.

To determine the lipid contents of the RNAi transformants, the Nile red fluorescence method was applied according to Chen [52 (link)]. Briefly, the cells were resuspended in 200 μL of staining solution containing 25% (v/v) DMSO and 0.5 μg mL^-1 Nile red dye for 10 min, and then fluorescence detection (FD) was performed using a Glomax-Multi Detection System (Promega), with excitation and emission wavelengths of 530 nm and 575 nm, respectively. Triolein (Sigma) was used as the lipid standard. The cell density (numbers/L) was determined using a cell counting method. The lipid content (ng/10⁶ cells) was calculated using the following equation: [0.0004×FD(530/575)-0.0038]×0.05/cell numbers.
For microscopic analyses, the cells were stained with Nile red (10 g/m³ final concentration), and the images were acquired using a Nikon 80i fluorescence microscope. Nile red signals were captured at an excitation wavelength of 480 nm, and the emission was collected between 560 and 600 nm.

Cells from neurosphere differentiation cultures were fixed in 4% PFA with a 30% sucrose solution for 30 min at 37°C. For immunocytochemistry, cultures were preincubated for 1 h in blocking solution (10% goat serum, 0.1% Triton X-100, BSA), followed by overnight incubation with the appropriate primary antibody at 4°C. The following primary antibodies were used: mouse anti-hGFAP (1:500, Sternberger Monoclonal), chicken anti-vimentin (1:200, Millipore), rabbit anti-NG2 (1:200, Millipore), mouse anti-Olig2 (1:200, Millipore), chicken anti-Tuj1 (1:200, Millipore), rabbit anti-active caspase-3 (1:200, Abcam), rabbit anti-HSP27 (1:200, Abcam), and rabbit anti-cathepsin (1:200, Abcam). The corresponding secondary antibodies were incubated for 2 h (Alexa-Fluor 405, 488, 555, or 647 goat anti-mouse, chicken or rabbit; 1:500; Invitrogen), followed by incubation with DAPI (1:1,000, Sigma) for 10 min and rinsing before being mounted on glass slides with Fluorsave (Calbiochem). Analyses were performed with a Nikon 80i fluorescence microscope at 40× or 63× magnification.

Bisbenzimide (Hoechst 33342; Sigma-Aldrich) was used here as a live cell DNA stain probe to highlight the formation of apoptotic bodies during cell apoptosis. The buffalo DCs treated with or without FgESPs for 48 h were harvested and transferred into a round-bottom Eppendorf tube in triplicates. Hoechst 33342 was added to the tube according to the manufacturer’s instructions, and incubated at room temperature in the absence of light for 20–30 min. After centrifugation at 1500× rpm and two washes in cold PBS, cells were made into smear slides, and observed under a Nikon 80i fluorescence microscope (Nikon, Milton Keynes, UK) with a digital camera attached to the computer. The apoptotic cells were then quantified by the mean percentage of Hoechst-positive nuclei (blue color) per optical field from at least 10 fields. Giemsa staining was also performed to display the microscopic structure of the cells. The cells were collected, washed, and fixed to the slide with 4% paraformaldehyde (PFA) solution. After air-drying for a few minutes, Giemsa stain solution (10× stock solution; Solarbio, Beijing, China) diluted in PBS (v:v = 1:9) was added dropwise to the stained cells. The slide was dehydrated, vitrified by xylol and mounted in neutral balsam. The microscopic structure of the stained cells was observed and photographed under a light microscope.

In our quest to assess cellular hypertrophy, we adopted the RP staining method. In the initial phase, primary cardiomyocytes underwent fixation using a 4% paraformaldehyde solution, after which we permeabilized them with 0.1% Triton. Following this, we proceeded to stain these cells with phalloidin–rhodamine at a dilution ratio of 1:200. In the concluding steps of the procedure, we used 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China) for counterstaining. We meticulously examined the stained cells and captured their images using a Nikon 80i fluorescence microscope (Nikon, Tokyo, Japan), thus enabling us to closely observe and analyze cellular hypertrophy in these primary cardiomyocytes.

Mouse PaSCs from adherent cultures were digested with 0.25% trypsin/EDTA and centrifuged at 800 rpm for 3 min. The cell pellets were resuspended in complete medium. After preparing 6-well plates with coverslips, cell suspension was added into each well. The cells were cultured at 37 °C in 5% CO₂ for 48 h, washed with PBS and fixed with 4% paraformaldehyde (PFA) (ZSGB-BIO, Beijing, China) for 15 min. Then, cells were treated with 10% donkey serum at room temperature for 1 h and incubated with the following primary antibodies: mouse monoclonal anti-α-SMA (1:100), rabbit polyclonal anti-fibronectin (1:50), and rabbit polyclonal anti-collagen I (1:50) at 4 °C overnight. The cells were then incubated with Alexa 594-conjugated anti-rabbit IgG (Invitrogen, Chicago, USA) (1:1000) or Alexa 488-conjugated anti-mouse IgG (Invitrogen, Chicago, USA) (1:1000) for 1 h at room temperature. Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Munich, Germany) for 5 min. The stained coverslips were visualized using a Nikon 80i fluorescence microscope.