Fv10 asw 2

Silver grain stainings of in situ hybridization were examined using an Axioskop2 microscope and photomicrographs were taken with Axiovision software (Zeiss, Jena, Germany). Images of immunofluorescence stainings were obtained with a laser-scanning confocal microscope (IX81-FV1000, Olympus, Tokyo, Japan) using Olympus FV10-ASW 2.0 software. All images were processed with the FIJI software (Schindelin et al. 2012 (link)). Representative images were adjusted for brightness and contrast. Overview figures were created from 16 to 30 single images using the MosaicJ plugin (Thévenaz and Unser 2007 (link)). The different anatomical brain regions were identified with the mouse brain atlas (Paxinos and Franklin 2008 ). Signal intensities were rated into one of the following categories: − not detectable, + weak signal, ++ moderate signal and +++ strong signal, according to the grey-intensity levels in the autoradiograph and also the expression observed in the high-resolution photomicrographs of [³⁵S]-labeled DRR1 mRNA. Densitometric quantification of autoradiographs of [³⁵S]-labeled mRNA was done using the Fiji software and grey values were expressed as arbitrary units (a. u.). Four different brain slices/ animal were measured. Background signal was subtracted outside the tissue section.

Stably transduced HCT116 tumor cells expressing the GFP-LC3 gene were generated by lentiviral transfection of the pCT-autophagosome-GFP Vector (Lentiviral Laboratory of the Vector Core Facility of the University of Pittsburgh) and selected with puromycin. HCT116 cells were treated with 10 μg/ml melphalan, along with 50 nM bortezomib and/or 2.5 μg/ml rapamycin for 24 h. Cells were washed three times with PBS, followed by fixation in 4% paraformaldehyde for 15 min. Nuclei were stained with DAPI (Cell Signaling). Slides were mounted and visualized in 0.4 μm sections using an OlympusFluoview 1000 confocal microscope and the companion software FV10-ASW2.1 (Olympus, Center Valley, PA, USA) under a × 63 oil immersion objective. GFP-LC3 puncta formation was quantified by counting at least 300 cells for each sample and plotted as mean±S.D. of three independent experiments.

The experiments were done essentially as described elsewhere [16 ]. Briefly, clots were formed inside the eight wells LabTek chambers (Invitrogen, Nalge Nunc International, Rochester, NY). Plasma samples were mixed with Alexa Fluor 488-labeled fibrinogen (10 μg/315 μl final sample volume), then clotted with thrombin-CaCl₂ solution (0.3 U/ml and 20 mM, respectively, final concentration). The clots were left for 2 h in a moist environment at 37 °C in order to fully polymerize.
The fibrin clots were observed in a Nikon Eclipse TE 2000 U laser scanning confocal microscopy (LSCM), with an argon ion laser (473 nm excitation and 520/540 nm for emission). The objective used was Plan APO VC 60X water immersion with a work distance of 0.27. The acquisition pinhole was set to 60 μm. Image analyses were done as described [21 (link)]. A z-stack of 60 slice was use for construct a 3D projection of 30 μm thick (0.5 μm/slice) were done. Five image by clot (212 × 212 μm) for each experiment (control and patient) were accomplished. Two diagonal lines, a horizontal and a vertical were drawn on the volumetric image of the stack using the Olympus FV10-ASW 2.1 software for obtain the pseudocolor perfil by line. Line graphs were used to calculate density (picks/μ) and diameter of fibers (μm) with Origin Pro 8 software.