Iplab software

IF was conducted using thin sections of human pancreatic fragments and acinar cell-blocks. Briefly, the sections were deparaffinised with xylene following which they were washed and fixed in 100% methanol and incubated with primary antibody (1:1000) at 4 °C overnight, followed by fluoroscent tagged secondary antibody (1:2000) in dark at room temperature. After several steps of washing, final incubation with DAPI for 15 min was performed. At least 5 randomly selected images per histology sections from each experiment (n = 3) were viewed in the Olympus IX71 fluorescence microscope, and fluorescent images captured using the CARVII bioimager (BD Biosciences) mounted with the IPLAB software (BD Biosciences). We quantified IL-6 or TNF-α positive cells per 100 amylase positive cells and expressed as mean (SEM).

An Olympus IX81 ZDC inverted microscope was used to acquire phase contrast images of the spheroid both before and 36 hours after mechanical stimulation was applied. Live spheroids were imaged at 37°C with 5% CO₂ within a custom stage incubator. Images were captured using a 10x/0.25NA CP-Achromat lens and SPOT Boost EM-CCD-BT2000 camera (Diagnostic Instruments) driven by IPLab software (BD Biosciences). The distance that cells disseminated from the spheroid was measured by drawing a line from the spheroid edge to the furthest cell disseminated using ImageJ (NIH).

Young panicles of at meiosis stage were harvested and fixed in Carnoy's solution (ethanol:glacial acetic acid = 3∶1) for chromosome spreading. Meiotic chromosome preparation and immunofluorescence were performed as previously described [34] (link). The FISH procedure was performed as described [60] (link). Microscopy was conducted using a ZEISS A2 fluorescence microscope with a microCCD camera. Image capture and analysis was carried out using IPLab software (BD Biosciences).

An upright Olympus B64 microscope with an attached short arc mercury lamp as an excitation light source was used. The excitation source and fluorescence emission are filtered through a filter cube (Ex. 577/10×, Em. 620/60 m, dichroic 585LP) from Chroma Technology Corp, Bellows Falls, VT. The emission light was detected by a CCD camera (Sensicam QE, Cooke Corporation, Romulus, MI) and captured images were analyzed using IPLab software (BD Bioscience, Rockville, MD). The setup used to seal the assay solution within fiber wells is described elsewhere [18] (link) and is shown in Figure S1A in File S1. In addition to the previous setup, a flat Peltier heating plate with an attached thermocouple was installed between the heat sink and the PDMS sheet. The Peltier plate and thermocouple are controlled and recorded by a computer via LabView (National Instruments, Austin, TX). The heating plate was calibrated prior to each experiment. The temperature variations within a single experiment did not exceed more than 0.5°C. By changing the current (3.0 A), voltage (4.0 V) and pulse duration (3 sec) it was possible to heat a sample to 47°C in 4 sec and cool it back to 20°C within 20 sec (Figure S1E in File S1).

BM-derived epithelial and stromal cells were counted by systematically examining the slides under ×40 magnification using an Olympus BX-51 microscope (Olympus). Areas of endometrium were randomly chosen and full-thickness counted. Multiple random slides of uterine sections from each mouse uterus were stained for use. Photomicrographs were taken using an Olympus BX51 fluorescence microscope. Images were captured with a digital camera, using IPLab software (BD Biosciences, www.bdbiosciences.com). Images were obtained using appropriate excitation and emission filter sets for 4′,6′-diamidino-2-phenylindole (nuclei), rhodamine (Y chromosome), fluorescein isothiocyanate (CD45, F4/80), and Cy5 (cytokeratin and vimiten). For quantitative trichrome analysis, five random sections of trichromatic slides were electronically scanned into an RGB image at magnification of 400X, which was subsequently analyzed using Image J (version 1.32j) software (National Institutes of Health, USA http://rsb.info.nih.gov/ij/). The amount of fibrosis (density and area) was then estimated from the RGB images. [15] (link) Quantitative of trichromatic sections were assigned in a double-blinded way by two people. Data presented are mean ± standard error of the mean. The Mann-Whitney Rank Sum test was used to compare the data.