Metamorph imaging software

Cells cultured on glass coverslips were fixed in 4% paraformaldehyde for 15 min. After permeabilization within 0.1% Triton X-100 in PBS for 20 min, cells on glass coverslips were incubated with primary antibodies overnight at 4°C, and then incubated with fluorescence-labeled secondary antibodies (Life Technologies) for 1 h at room temperature. Stained samples were mounted with ProLong Gold Antifade Reagent with DAPI (#8961, Cell Signaling Technology). Samples were observed using an Eclipse TE2000-U inverted microscope (Nikon) driven by Metamorph imaging software (Molecular Devices). The images were analyzed using NIS-Elements Advanced Research software (Nikon, version 4.50).
For tissue immunofluorescence, all collected fresh samples were fixed in 4% paraformaldehyde followed by dehydration, paraffin wax embedding. 5 μm paraffin sections were made and microwave repair was performed for antigen retrieval. Afterward, sections were incubated with a primary antibody against CD8 (Cell Signaling Technology, Catalog Number: 85336) in 2% BSA. After incubation with second antibodies, all sections were covered with coverslips with mounting medium containing DAPI. All specimens were observed under a fluorescence microscope (Nikon). The degree of CD8⁺ T cell infiltration was measured with 10 independent high-power microscopic fields for each tissue sample (n = 20).

Cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed in PBS, and labeled with a mouse monoclonal antibody to βIII tubulin (1:900 for DRG; Promega), followed by a 45 min incubation in an AlexaFluor 488 labeled secondary antibody (1:400, Life Technologies, Grand Island). Coverslips were placed onto slides with FluoroGel (Electron Microscopy Sciences). Fourteen-bit images were collected on a Leica DMI 6000 B inverted microscope (Leica Microsystems; Wetzlar, Germany) using a Retiga Aqua blue camera (Q-imaging; Vancouver, British Columbia). Briefly, the entire coverslip was imaged at 10x and all resulting images were stitched together using the scan slide function in MetaMorph Imaging Software (Molecular Devices) to generate a full resolution composite image. The composite image was then subjected to neurite outgrowth analysis using the neurite outgrowth module in MetaMorph software. The longest neurite from each βIII tubulin-positive neuron with a process of at least 1.5 times the diameter of the cell body was measured.

Hearts were excised and fixed in 4% paraformaldehyde, and then embedded in paraffin. Transverse sections of heart were cut in 4μm sections at the papillary muscle level and subsequently stained with hematoxylin and eosin, Masson-trichrome, and Picrosirius red. Approximately 10 visual fields on two randomly selected sections from each animal were visualized by light microscopy using an objective with a calibrated magnification (×400). Perivascular fibrosis (area of Picrosirius red-positive staining surrounding the vessel/total lumen area) in LV wall and interstitial fibrosis (area of interstitial Picrosirius red-positive staining/total myocardial area of the field) in papillary muscle, were quantified by MetaMorph imaging software (Molecular Devices, Sunnyvale, CA).

3% w/v agarose gel pads were made using a low-melting-temperature agarose (SeaPlaque, Lonza) and EZRDM media. 1.5mL of cells were harvested by spinning down in a bench-top microcentrifuge at 4.5 rcf for 5 minutes and resuspended in ~100μL of fresh EZRDM. Cells were pipetted onto agarose pads and sandwiched between the agarose pad and a #1 coverslip. Immobilized cells were imaged on an Olympus IX71 inverted microscope with a 100x oil objective (PN, NA =1.45) with 1.6x additional magnification. Photons from cells were collected with an Andor EMCCD camera using MetaMorph imaging software (Molecular Devices). Fluorescence from cells was obtained using solid-state lasers at 405nm and 568nm wavelengths (Coherent). All SMT images were collected using 5ms exposure with 1.74ms of cycle time for a total frame length of 6.74ms. Three movies consisting of 2500 frames each were taken for each cell. No cell was imaged longer than five minutes to avoid phototoxicity effects. Activation of the fluorescent proteins was continuous throughout imaging, and no changes were made to either the activation or excitation power throughout the imaging session.

Cells were grown to approximately 80% confluence on 4-well chambered slides and fixed with warm 3.7% formaldehyde for 10 min at room temperature. Cells were permeabilized after rinsing thoroughly in 0.2% Triton X-100 for 5 min and rinsed 3-4 times with PBS. Cells were incubated with approximately 200 uL of FX Signal enhancer (Invitrogen) for 30 min in a humid environment and at room temperature. Cells were stained after rinsing thoroughly with anti-Kaiso antibody for 1 hour at room temperature, protected from light. Cells were washed thoroughly and stained with secondary antibody, Alexa 594 anti-mouse (Invitrogen) for 30 min and washed thoroughly. Nuclear staining was achieved by adding Vectashield mounting medium with DAPI and cell staining was observed under DSU confocal microscope (Olympus, New York) using Metamorph Imaging Software (Molecular devices, LLC, Sunnyvale, CA) to capture cell images.

For tumor sphere formation assays, 1000 cells per ml were plated in 2 ml in 6-well ultra-low attachment plate (Corning) in MammoCult medium (Stem cell Technologies) in the presence of amiodarone. After 7 days, 10 × 10 stitch imaging was done at 10× (100 random images acquired) using a Retiga Aqua Blue camera (Q Imaging, Vancouver, BC) connected to a Leica DMI6000 inverted microscope. Individual images were taken and then a composite image was generated using the scan slide function in MetaMorph Imaging Software (Molecular Devices, Downingtown, PA). Subsequent integrated analysis also used MetaMorph software.