Bx51 upright epifluorescence microscope

Cells or tissues were stained per the manufacturer’s protocol using rabbit polyclonal (ab19857, Abcam, Cambridge, MA; 1:200 dilution) or murine anti-OCT-4 (ab91194, Abcam, Cambridge, MA; 1:200 dilution), and rat anti-Vimentin (ab115189, Abcam, Cambridge, MA; 1:200 dilution). Secondary antibodies were Allophycocyanin anti-mouse IgG (A-865, Life Technologies, Grand Island, NY; 1:1000), AlexaFluor 555 anti-rabbit IgG, and anti-Rat IgG AlexaFluor 647 (Abcam, Cambridge, MA; 1:1000 dilution). Cells were visualized with an Olympus FV1000 Confocal Microscope using 488 nm, 543 nm, or 633 nm for excitation. Tissue was visualized with an Olympus BX51 upright epifluorescence microscope (Olympus America, Center Valley, PA) using excitation/emission filters of 480 nm/535 nm, and 620 nm/700 nm.

The effect of ASK1 inhibitor on cell migration was measured using wound healing or scratch assay. Briefly, 2×10⁵ cells were seeded in a 6 well plate and incubated for 24 h and then cell monolayer was scraped with a 200 μl pipette tip in a straight line to make a wound. Thereafter ASK1 inhibitor TC ASK 10 at 100 nM was added and incubated for a further 12 h. Images were taken with Olympus BX51 upright epifluorescence microscope fitted with a DP70 CCD camera (Olympus, Shinjuku, Japan) and analyzed using the ImageJ software by measuring the % cell-covered area of the wound.

Imaging was performed on an Olympus BX51 upright epifluorescence microscope using an Olympus PlanApo ×63 oil objective for chromosomes and ×40 air objective for nuclei and spindles. Images were captured on a Hamamatsu ORCA-II camera. In order to ensure that fluorescence intensity values were comparable between samples, exposure times for each channel were set by the sample with the highest fluorescence intensity and kept constant for all replicates of that experiment.

Computer-assisted image analysis was performed with a NanoZoomer-SQ Digital slide scanner (Hamamatsu, Hamamatsu City, Japan), Olympus BX51 upright epifluorescence microscope fitted with a DP70 CCD camera (Olympus, Shinjuku, Japan) and Image-J software. Prior to image analysis observer was blinded to subject and diagnosis. After a minimum of 6–7 images per tissue were taken, then four were randomly selected per patient to be quantified. The percentage staining was quantified in the airway wall and in the ASM bundles using Image-J software.

All the animal experiments were conducted in accordance with the approved guidelines for the care and use of laboratory animals in research, and the protocol was approved by the Washington University Animal Welfare Committee. BxPC-3, MDA-MB-231, and EMT-6 cells were injected subcutaneously into the dorsal flanks of athymic nude mice, while 4T1luc cells were injected into Balb/c mice. Mice were sacrificed when the tumors were between 0.5 and 1.5 cm in diameter. In certain cases, tumors may have had exposure to the near-infrared tumor imaging agent cypate prior to excision. Tumors were excised based on gross appearance and frozen in OCT media. The average pH in 4T1 tumor models was 6.8+/− 0.1 pH units, as measured by an invasive probe (5 mm probe). The average pH level in EMT-6 and BxPC-3 tumors was not measured, previous studies reported acidity in BxPC-3 and EMT-6 tumor models and the importance of the pH for chemotherapy resistance19 (link). Tissue sections were cryosectioned at 10 m thickness. Tissue was visualized with an Olympus BX51 upright epifluorescence microscope (Olympus America, Center Valley, PA) using excitation/emission filters of 480 nm/535 nm, and 620 nm/700 nm.

Images captured with the Nikon A1 Confocal microscope and those generated by the DeltaVision OMX 3D-SIM were processed using Bitplane, Imaris Scientific 3D/4D image processing software to create Maximum Intensity Projection (MIP) and slices images.
M. hyopneumoniae-infected PK-cells and uninfected controls were labelled with integrin β1 (described above) and 10 random fields of view were captured using an Olympus BX51 Upright Epi Fluorescence Microscope at 20 × magnification and a constant exposure. The mean fluorescence of integrin β1-stained samples was calculated, after thresholding and binary conversion, using ImageJ. GraphPad Prism 7 was used to plot the data, including the standard error of the mean, and to perform the statistical analyses (unpaired t-test).