Cellsens dimensions software

For each rat, a small gastric tissue section was fixed with 10% neutral buffered formalin, embedded in paraffin, cut into 5 μm thickness and stained with hematoxylin and eosin stain. The specimens were examined under Olympus BX43 light microscope, and sections were captured by Olympus DP27 camera connected to Cellsens dimensions software (Olympus). A 0–14 range was used to score the microscopic damage according to [53 (link),54 (link)], where epithelial cell loss or the presence of inflammatory cells scored 0–3 and oedema in the upper mucosa or hemorrhagic damage scored 0–4. The total microscopic score was obtained by summating the four histopathological scores.

For tube formation endothelial cells were grown on Matrigel™ (BD Biosciences, following standard methods [27 (link)]. Tube number and length were analyzed from randomly selected fields. For wound healing a scratch (wound) was made across a cell monolayer using a sterile tip. Image analysis was conducted using ImageJ software. Relative clearance rate was determined using the equation: (distance_=0h – distance_=24h)/ distance_=0h × 100 % [27 (link)]. For migration assays, we used a transwell 8 μm polycarbonate membrane assay (Corning, Tewksbury, MA), with either conditioned media, or recombinant purified CCL5/RANTES (10 nM) (PreproTech, Rocky Hill, NJ) as chemoattractant. After four hours membranes were imaged using the IX71 fluorescent microscope and counted using CellSens Dimensions software (Olympus, Tokyo Japan).
Endothelial cells were transfected with siRNAs (0.04 pmol/μl) using siPORT NeoFX Transfection Agent (Ambion, Carlsbad, CA) [27 (link)], and/or treated with the small molecular inhibitor of CCR5, maraviroc (100 nM). Pooled siRNAs specifically designed to inhibit mouse or human CCR5 (siGENOME SMARTpool) and Cyanine 3 (Cy3) labeled siGLO RISC-Free Control siRNA were obtained from Thermo Scientific (Waltham, MA).

Anthers at varying stages of development were fixed in 6% glutaraldehyde and 4% paraformaldehyde in 50 mM sodium phosphate buffer (pH 7.5) for 2 h at 4°C followed by 15 min of vacuum infiltration (Li et al., 2007 (link)). Fixed tissue was dehydrated using the following ethanol gradient series: 30, 60, 70, 90, and 100%. Subsequently, samples were infiltrated with LR white resin/ethanol. Anthers were embedded overnight at 65°C and sectioned using a Reichert ultramicrotome (Reichert, Depew, NY, USA). Semi-thin transverse sections (3.0 μm) through the center of the anther were stained with 1% toluidine blue. Sections were viewed with brightfield conditions using an Olympus BX53 Upright Microscope, captured with the Olympus cellSens Dimensions software, and photographed with an Olympus DP80 Color microscope camera (Olympus, Shijuku, Tokyo, Japan).

The frozen cardiac tissue of ACT patients, as well as healthy controls, was embedded, sliced, and stained with Masson’s trichrome staining in cooperation with the Department of Pathology of the University Medical Center Göttingen. The area of fibrosis was quantified in cooperation with the Technology Platform Clinical Optical Microscopy. The slides were first scanned for virtual microscopy using a 20 × objective (UPlanApo) with the dotSlide SL slide scanner (Olympus) and a peltier-cooled XC10 camera. Fibrotic areas were then calculated by a scientist blinded to analyzed myocardium, using the CellSens Dimensions software (Olympus).

Hearts were perfused ex vivo via the aorta with 0.9% NaCl before fixation in 3.7% formaldehyde solution overnight. After embedding in paraffin, 5 μm sections were cut and stained for 60 min at room temperature in Sirius Red solution, i.e. 0.1% Direct Red 80 (Sigma, Taufkirchen, Germany) in saturated aqueous picric acid (Morphisto, Frankfurt, Germany) adjusted to pH 2.0 with sodium hydroxide [24 (link)]. The slides were tipped four times in 0.5% acetic acid solution and incubated 5 min each in ethanol and in isopropyl alcohol. After two further incubations for 10 min in xylole, the slices were embedded in Histokitt (Carl Roth) and recorded at 20x magnification using a slide scanner (Olympus, Hamburg, Germany). The extent of fibrosis based on the amount of collagen was quantified in two complete heart sections using cellSens Dimensions software (Olympus) by a scientist blinded to the genotype of the mice.
Kidneys were immersion-fixed in 3.7% formaldehyde solution overnight and embedded in paraffin. Immunohistochemical demonstration of eGFP was performed on 5 μm sections using a rabbit anti-eGFP polyclonal antibody (Abcam, Cambridge, UK) and the Vectastin ABC kit (Vector Laboratories, Burlingame, CA) using DAB (3,3’-diaminobenzidine tetrachloride) as a substrate. Quenching of endogenous peroxidase activity was achieved by treating the sections with 3% H₂O₂ in ethanol.

Caspase-3 protein was localized within the cytosol of necrotic neurons using a casp-3 primary antibody and DAP Kit. Formalin‐fixed paraffin‐embedded sections were incubated with casp-3 primary antibody (Abcam, Ltd.) at 1/200 dilutions, then incubated with Peroxidase Block (Sakura BIO) and the reagent required for the identification of the antigen‐antibody complex (Power‐Stain 1.0 Poly HRP DAP Kit; Sakura). The sections were treated with 3,3′‐diaminobenzidine chromogen substrate for 10 min and counterstained with Hematoxylin and inspected by a light Olympus BX43 microscope, and caught images by Olympus DP27 camera connected to CellSens dimensions software (Olympus).

De-paraffinized and hydrated sections from hepatic and renal organs were rinsed with PBS for 15 min. The sections were blocked with normal goat serum (1.5% in PBS) and then incubated (45 min, room temperature) with HIF1-alpha antibody specific for the active form (1.0 μg/ml). Then, the sections were incubated with gold (1 nm)-conjugated goat anti-rabbit IgG (1:200; 30 min, room temperature) and developed with silver enhancement solution (Amersham Pharmacia Biotech silver enhancement system) for 5 min. Sections were counterstained with methyl green. For negative controls, rabbit IgG (1 μg/ml) instead of the primary antibody was added to the reaction. Finally, the tissue sections were counterstained with hematoxylin and mounted using DPX and were examined using light microscope (Olympus, CX41, Japan). The percentage of surface area expressed by HIF1-alpha in liver and kidney sections from different groups was assessed using 3 sections for each slide. At least 15 fields per section were accounted using CellSens dimensions software (Olympus).