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CHAPTER 3 To detect cells undergoing apoptosis/necrosis, a TUNEL assay was performed. Slides were incubated with 1:10 Terminal Deoxynucleotidyl Transferase (TdT) buffer (125 mM Tris-HCl, 1M Sodium Cacodylate, 1.25 mg/ml BSA, pH 6.6) for 10 min and then 1hr incubation at 37 °C with reaction mixture [0.5 enzyme unit/μl TdT (Roche Applied Science), and 2.52μM Biotin 16 dUTP (Roche Applied Science) diluted in 1:10 TdT buffer]. This was followed by 15 min incubation in 1:10 saline Sodium Citrate (SSC) buffer (175.3 mg/ml Sodium Chloride, 88.2 mg/ml Sodium Citrate, pH 7.0) and blocked with 10% normal goat serum in 0.1M PBS for 10 min before incubating with secondary antibody in 0.1M PBS (1:1000 dilution, Invitrogen, Alexa 488 conjugated streptavidin S11223) at 37 °C for 30 min. All image analysis was performed blind to the experimental group. 2D images were constructed from three colour channel (red, green and blue) images acquired from a LED fluorescent microscope (Carl Zeiss Colibri) with a 20× objective and digital camera (AxioCam MRc 5) with all settings kept constant for each channel. Cells with co-labelling were quantified with ImageJ (v1.46r) using the Cell Counter plugin that enables the placement of different classes of markers onto an image. Cytoplasmic markers, a class for each channel, were used to tag positive label in a single focal plane for all green and red channels that were examined independently. To define ED1+ cells, the accompanying DAPI+ nucleus (blue channel) was tagged for cells where ED1 staining was clearly complementing the DAPI surface profile. Double labelled cells (i.e. ED1+Arginase1+ or CD80+) were evaluated by scrutinising all tagged DAPI+ cells for co-labelling in red and green channels. These cells were tagged again with another marker class. All markers were automatically quantified for each class by the software. Cells out of focus were not included. Cell counts were obtained from dorsal horn regions with viable tissue and quantified as the mean of duplicate images, each covering a minimum area 0.05 mm2. The areas of interest were defined and quantified prior to cell quantification, and included the dorsal horn grey matter region as well as the white matter in the surrounding dorsal columns and lateral funiculus. Cell quantification is expressed as the number of cells per unit area (mm2). 3.3.9 Statistics All data expressed as box plots and individual points in figures or as mean ± SEM in the main text unless otherwise stated. Box plots indicate the median, upper and lower quartiles with whiskers extending to maximum and minimum values excluding outliers (more than 1.5 times respective quartiles). Statistical analysis was carried out using R or MATLAB, and a criterion alpha level of 0.05 was adopted as statistically significant. Data sets were tested for normality and homoscedasticity and t-tests, ANOVA, linear mixed models were applied for normally distributed data (indicated by *) and Wilcoxon rank-sum (indicated by †) where data was not normally distributed. 62PDF Image | Effects of Red Light Treatment on Spinal Cord Injury
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