Imagej software

An aliquot of 40 μL running buffer (10 mM Tris-HCl, 0.05% v/v Tween 20, pH 7.0) was mixed with 0.1μL of the AuNPs-biotinyl-αIL6ab conjugates, and the solution mixture was then applied to the streptavidin coated LFIDs and GFC-LFIDs and allowed to run for 10 min. The assay was completed by further addition of 20 μL of the running buffer. For quantitative evaluation, images of the GFC-LFIDs were recorded using a flatbed scanner (Epson Perfection V370 Photo). The color intensities of the test zones were analyzed with ImageJ software (Scion Corp., USA) and quantified as a function of pixel intensity.

The nanofibers were gold-coated, as described by Heunis et al. [13 (link)] and the surface structure of the nanofibers studied using a scanning electron microscope (SEM, Leo 1430VP, Zeiss, Cambridge, England). From these images, the diameter of the nanofibers was calculated using ImageJ Software, version 1.46, Scion Corporation [14 (link)]. Protrusions from the nanofibers were visualised by transmission electron microscopy (TEM), using a Philips Tecnai TF20 (FEI, OR, USA). Surface topology was studied using a Nanosurf atomic force microscope (AFM) Easyscan 2 (Nanosurf Inc., CA, USA). Interactive properties of CIP, PDLLA and PEO were studied using a Fourier transform infrared (FTIR) spectroscope (Thermo Nicolet Avatar 330, Thermo Scientific, Waltham, MA, USA), equipped with a Smart Performer Zn/Se ATR (attenuated total reflection) accessory. Crystal formation and phase compositions were observed by X-ray diffraction (XRD), using a Bruker AXS D8 Advance X-ray diffractometer (Bruker AXS, Frankfurt, Germany) operated in locked coupled mode. The instrument was equipped with a Vantec-1 position sensitive detector optimized for Cu-Kα radiation at λ = 1.5406 Å. The X-ray tube was operated at 40 mA and 40 kV. Readings were recorded at a scanning rate of 1 sec/step, with a step size of 0.0275° in a 2θ range that extended from 4° to 69.99°.

The mitochondrial ultrastructure of the soleus and gastrocnemius muscles was evaluated using TEM as previously described [35 (link)]. Images were taken at 10.000-fold magnification and ten random pictures from both muscles of each mouse were analyzed using the ImageJ software (Scion Image, National Institutes of Health, Bethesda, USA). Mean mitochondrial size was determined by manually encircling all identified mitochondria. Alterations on mitochondrial architecture were assessed by the categorization “normal” or “damaged” as follows: Mitochondria showing a loss of more than 50% of the cristae or a disruption of more than 50% of the outer membrane were assigned to “damaged” or otherwise to “normal.” The results were given as the percentage of the total number of mitochondria. Moreover, the percentage of swollen mitochondria and mitochondria containing multi-lamellar bodies was determined.

A wound healing assay was used to measure cell migration capability. Both Du-145/R and PC-R were plated in 6-well plates. Afterwards, a sterile 200-μL pipette tip was used to scrape a vertical wound. The cell debris was removed by washing with PBS twice and the cells were treated with serum free RPMI 1640 medium containing the following: vehicle control (DMSO), 20 μM Que, 1.0 nM Doc, and 20 μM Que + 1.5 nM Doc. The assays were performed in triplicate, and at the end of the incubation period, images were obtained with an inverted microscope at base line and on termination (48 h). The images and the area of wound healing were calculated by Image J software (Scion Corp., Frederick, MD, USA).

Cultured media harvested from cells were analyzed for MMP2 and MMP9 by gelatin zymography, as described previously [25 (link)]. Briefly, conditioned media aliquots were resuspended in nonreducing sample buffer and applied to 10% SDS-PAGE copolymerized with gelatin (1 mg/mL). After electrophoresis, the gels were washed for 1 h in Triton-X-100 (2.5% v/m), incubated overnight in an enzyme buffer (developing buffer) at 37 °C, stained in Coomassie solution for 1.5 h and subsequently destained for 1 h in a destaining solution. By that, the enzymatic active areas became visible as a transparent band on the blue-stained gel. The zymograms were analyzed with Scion ImageJ software.