Eos rebel xsi camera

The assay was performed by plating in triplicates 1×10⁵ OVCAR3 or OVCAR3-sfRon cells per well of 96-well plate in serum deprived medium (0.5% FBS in RPMI 1640). Cells were incubated for 2h at 37°C followed by washing away unattached cells. Adherent cells were fixed with ice-cold methanol for 10 min and then stained with 0.5% crystal violet solution (made in 25% methanol and stored at room temperature) for 10 min in room temperature. Next, culture wells were rinsed with ddH₂O until purple color was no longer coming off while rinsing and culture plates were dried overnight. Dried culture plates were imaged on an Olympus Bx50 microscope with a Canon EOS Rebel XSI camera using EOS imaging software and quantified at OD 595 nm after extraction using FLUOstar Omega Microplate Reader (BMG LABTECH, Cary, NC)

Two mice bearing PDX-0113 from each group were sacrified 3h after drug(s) administration. Harvested tumors were fixed in 10% neutral buffered formalin, paraffin embedded, and hematoxylin–eosin (H&E) stained according to our standard protocols [7 (link), 9 (link)]. Tumors were analyzed by IHC for expression of the following markers: anti-human cytokeratin (1:400, DAKO #Z0622), PAX8 (1:1000, Abcam, #ab189249), WT1 (1:1250, Cell Signaling, #83535), phospho-S6 (1:200, Cell Signaling, #4856), Ki67 (1:200, Thermo Scientific #RM-9106-S1) or cleaved caspase-3 (1:250, Cell Signaling #9661). Staining was visualized by 3,3-diaminobenzidine (DAB), with hematoxylin as a counter-stain. Slides were imaged on an Olympus Bx50 microscope with a Canon EOS Rebel XSI camera using EOS imaging software. For Ki67 or cleaved caspase-3 quantification, 3 images per tumor from two different animals from each treatment group were analyzed with ImageJ software [51 ]. The relative proliferation (Ki67) or apoptosis (cleaved caspase-3) were expressed as a number of positively stained nuclei in each image, as previously described [52 (link)].

After the staining procedures, photographs (40x) were obtained with a Primo Star Carl Zeiss trinocular microscope used for this purpose and a Canon EOS Rebel XSI camera with the software EOS Utility (version 2.4, Copyright^© CANON INC, USA, 2008) was used for image capturing. Later, they were quantified to determine the indirect presence of lipids as well as the degree of inflammation of the lesions, in terms of the presence of inflammatory cells; determining representative areas in each artery lesion with a dimension of 50 μm², the software AxioVision Release 4.8.1 (Carl Zeiss version 11. 2008) was used for this purpose, and the software Micrometrics SE Premium (version 2,8; ACCU-SCOPE Inc., Commack, NY, USA, 2008) was used for counting inflammatory cells. The lesion surface was quantified by using the ImageJ software (version 1,46j; National Institute of Health, USA, 2006) to determine the degree of fibrosis. Each image was analyzed through the Colour Deconvolution Plugin, selecting the vector for Masson's Trichrome, determining the quotient (density/area), and allowing the characterization of each lesion [23 (link)]. From each of the image analyses, 10 sections were measured by 2 independent operators (J.M. and W.D.).