Axio scope a1 fluorescent microscope

Morphological studies of H9c2 cardiomyoblasts to evaluate the magnitude of cell surface area (Pedram et al. 2005 (link)) were accomplished using TPP 24-well plates, in which ~ 8000 cells per well were cultured on glass coverslips. Cells were treated as described above, then observed using fluorescence microscopy. After 48 h following the hypertrophic challenge, cells were fixed for 15 min in 4% of formalin, washed three times and extracted in 0.1% of Triton-X solution for 15 min. Fluorescein isothiocyanate (FITC)-conjugated phalloidin was used to visualize cytoskeletal structures and 4′.6-diamidino-2-fenylindol (DAPI) stain for nuclear elements. The Zeiss Axio Scope A1 fluorescent microscope was used with ZEN 2012 software to acquire images and for measurements of cell area in square micrometers (μm²). Microscopy observations were expressed as the mean of four independent experiments, using 150 cells in each group.

Transwell migration assays were performed using 24-well polycarbonate Transwell inserts (3422, Sigma-Aldrich). C42B and 22RV1 cells transfected overnight with siRNA were seeded into the upper chamber of the Transwell at a density of 9.0 × 10⁴–1.5 × 10⁵ cells/well in serum-free medium. 650 μL of medium containing 10% FBS was added to the bottom chamber. Cells were incubated at 37 °C for 48 h. For TDZ treatment, medium in both the upper and lower chambers were supplemented with TDZ (2.5 μM). Inserts were washed with PBS and non-migrated cells were gently removed using a cotton-tipped swab. The inserts were then fixed in 4% paraformaldehyde for 20 min and stained with 1% crystal violet for 30 min. Images of migrated cells were captured using the Axio Scope A1 Fluorescent Microscope (Zeiss) at 40× magnification. The number of migrated cells was counted manually and presented as percentages relative to control cells ± SEM.

Cell invasion were assessed using 24-well-plate BD Biocoat Matrigel Invasion Chambers (In Vitro Technologies, NSW, Australia) according to the supplier instructions. After 48 hr of siRNA transfection, 650 µl of medium containing 10% FBS was added to the bottom chamber, and equal number of cells within 1% FBS-contained medium were transferred to the upper chamber. After incubation at 37°C, 5% CO₂ for 48 hr, non-invading cells as well as the Matrigel from the interior of the inserts were gently removed using a cotton-tipped swab. The inserts were fixed in 4% paraformaldehyde for 20 min and stained with 1% crystal violet for 30 min. The images of invaded cells were captured using the Axio Scope A1 Fluorescent Microscope (Zeiss) at 40X magnification. The number of invaded cells were counted manually and presented as percentages relative to control cells ± SEM.

FBs were fixed in 4% formaldehyde for 20 min at room temperature and then washed with 0.2% PBS in Triton (0.2% PBSTr). Following 1 h blocking with 10% heat inactivated goat serum in 0.2% PBSTr, FBs were incubated with Alexa 488 Phalloidin (Molecular Probes, 1:500) in block for 2 h at room temperature. Following a brief rinse, samples were incubated with DAPI (1 μg/ul) for 10 min at room temperature. Then samples were washed with PBS and mounted on microscopy slides with Vectashield and imaged on a Zeiss Axioscope A.1 fluorescent microscope.

For High-magnification observations of the plant samples with fluorescence, a Carl Zeiss AxioScope A1 fluorescent microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany) equipped with a Zeiss Axio Cam ICc1, with filter Set 43 or Rhodamine filter from Zeiss (Ex: BP 545/25, Em: BP 525/50) for red and green fluorescence, was used.
Low magnification images were observed under a Wild Heerbrugg stereoscope (Wild Heerbrugg Instruments, Ltd., Heerbrugg, Switzerland) using a Green –Only bandpass filter, Stereo Microscope Fluorescence Adapter SFA-LFS-GO (NIGHTSEA, Electron Microscopy Sciences, Hatfield, PA. 19440). Images were captured with a Canon PowerShot S3 IS (Martin Microscope Co., Easley, SC). The procedures and techniques described in this manuscript have been thoroughly described to allow for replication of the results. All relevant materials, steps, and parameters have been included to ensure full reproducibility of the experiment.

Transwell migration assays were performed using 24-well polycarbonate Transwell inserts (3422, Sigma Aldrich). LNCaP, 22RV1 and MR49F cells transfected overnight with siRNA were seeded into the upper chamber of the Transwell at a density of 8 × 10⁴–2.5 × 10⁵ cells/well in serum-free medium. 650 µl of medium containing 5% FBS was added to the bottom chamber. Cells were incubated at 37°C for 48 hr. Non-migrated cells were gently removed using a cotton-tipped swab. The inserts were fixed in 4% paraformaldehyde for 20 min and stained with 1% crystal violet for 30 min. The images of migrated cells were captured using the Axio Scope A1 Fluorescent Microscope (Zeiss) at 40X magnification. The number of migrated cells were counted manually and presented as percentages relative to control cells ± SEM.

BV2 and L929 cells morphology was evaluated by phase-contrast microscopy using a Primo Vert microscope and fluorescence images were captured using an Axio ScopeA.1 fluorescent microscope (Carl Zeiss MicroImaging GmbH, Gottingen, Germany). At least 8 images per condition were acquired using an AxioCam 105 Color camera with the Zen lite 2012 (both from Carl Zeiss MicroImaging GmbH). Quantification of p65 NF-κB signal was performed using ImageJ v3.91 software, by selecting a region of interest according to the localization of the nucleus and measurement of fluorescence intensity in the same region.