Q color 3

For the intracellular adriamycin delivery, A-NOA particles (1 mg/1 mL) were internalized into MCF-7 cells which are placed in a petri dish. The cells are removed from the petri dish after 30 min, and are washed three times with 1X PBS. Afterward, the cell is mounted on a microscope slide and a PDMS micro chamber was placed on the microscope slide. The microscopy system consisted of an inverted microscope (Olympus IX73) equipped with a dark-field condenser (1.2–1.4 numerical aperture) and a white light source (Xenon Arc Lamp) to acquire images. Then, using a digital camera, the dark images of the treated cell and A-NOA were acquired (Q-color3, Olympus). A monochromator (Acton Research) with a cooled spectrograph coupled charge device (CCD) camera was used to gather scattering spectra from the samples at different locations (Roper Scientific). In front of the monochromator, we set up a 2 μm-wide aperture to hold only a single probe in the region of interest.

After euthanasia, jejunum samples were removed and fixed in 10% buffered formalin for 24 h and then stored in 70% alcohol. Soon after, samples were dehydrated in alcohol and fixed in paraffin. The sections were stained with hematoxylin-eosin (H&E) for villus height measurement, which is a marker of intestinal tissue absorptive surface. Villus height was measured from the baseline to the top in clear transversal sections. Ten villi per animal were analyzed using an optical microscope (Olympus CX3, Japan) and an image acquisition system (Q-Color 3, Olympus). Measurements were taken from captured digitalized images using ImageJ 1.4^® software (NIH, USA) after proper calibration. All analyses were done blindly by an experienced histologist.

After albuginea removal, fixed testis samples were dehydrated in ethanol and embedded in glycol methacrylate (Leica Microsystems, Wetzlar, Germany). Three-micrometer-thick semiserial sections were obtained in a rotary microtome. To avoid analyzing the same histologic area, one out of every 20 sections was collected and used. Testis slices were stained with 1% toluidine blue/sodium borate [6 (link)]. Stereological estimations were obtained from digital images captured at different magnifications using a light microscope (Olympus BX-60, Tokyo, Japan) equipped with a digital camera (Olympus QColor-3; Tokyo, Japan) [29 (link)]. Stereological principles [30 (link)] were applied to investigate the general testis microstructure. The volume densities (Vv) (%) of seminiferous tubules, intertubules, blood vessels, lymphatic vessels, and connective tissue were estimated in 10 histological fields per animal (200x magnification) using a point counting method and the equation Vv = Pp/Pt, where Pp is the number of test points hitting the structure of interest and Pt is the total points in the test system (Pt = 266).

Ventricles were harvested following sacrifice and processed as previously described [9 (link)]. Ventricular transverse sections (8 μm) were stained with Masson’s trichrome to assess LV fibrosis. Macrophage content was detected by RAM-11 staining as previously described [10 (link)]. Aortic valves were opened longitudinally, and the three valvular cusps were separated. Left coronary cusps were immediately frozen in an embedding medium and stored at -80°C. The region of analysis (ROA) was composed of 1000 μm of the Valsalva sinus from the leaflet base and 500 μm of the leaflet from the leaflet base as previously described [12 (link)]. Haematoxylin-phloxin-safran (HPS) and von Kossa stained sections were prepared for plaque examination and tissue calcification, respectively and as previously described [10 (link),12 (link)]. Pictures were taken at 4X (HPS and Von Kossa staining on left coronary cusp), 10X (for RAM-11 and Sirius Red staining on LV sections) or 20X magnification (for Masson’s trichrome staining on LV sections) using a computer-based digitizing image system using a light microscope (Olympus BX41, Richmond Hill, ON, Canada) connected to a digital video camera Q-Color3 (Olympus, Richmond Hill, ON, Canada) using Image Pro Plus version 9.2 (Media Cybernetics, Bethesda, MD, USA) for picture acquisition and analysis (5 pictures/ LV section).

Histological analyses and LV morphometry were performed as previously described [37 (link)]. Briefly, LV sample fragments were collected, fixed in 10% formalin, dehydrated in ethanol, clarified in xylol, and embedded in paraffin. Blocks were cross-sectioned into 5 μm-thick histological sections and subsequently stained with hematoxylin–eosin (H&E) and mounted on histology slides. To avoid repeated analyses of the same histological area, sections were evaluated in a semi-series using one of every 10 sections. Slides were visualized, and images were captured using a light microscope (Olympus BX-50, Tokyo, Japan) connected to a digital camera (Olympus Q Color-3, Tokyo, Japan). Subsequently, analyses of the myocyte cross-sectional area (CSA), total collagen, percentage of cardiomyocytes, and extracellular matrix were performed.