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Albarracin et al. BMC Neuroscience 2013, 14:125 http://www.biomedcentral.com/1471-2202/14/125 was counted across the retina, from inferior to superior, and either expressed per unit area and/or total number of TUNEL + cells, in 3 sections per animal. Counts of TUNEL + cells in control animals were used to determine baseline levels of cell death. Results from 12 animals from each group were averaged and analysed. Immunohistochemical analyses Immunohistochemistry (IHC) was performed using stand- ard protocols for fluorescent imaging. Briefly, frozen sec- tions of the retina were placed in 70% ethanol followed by 2 rinses in 0.1 M of PBS. All sections were permeabilised with an antigen retrieval solution (Immunosolution Pty Ltd, Australia) for 45 mins at 37°C, followed by blocking with 10% normal goat serum (Sigma Aldrich, St Louis, MO, USA) for 1 h before incubation with the primary antibody for 24 h at 4°C. Primary antibodies used are mouse monoclonal antibody against cytochrome oxidase Va (1:100; Mitosciences, USA) and rabbit polyclonal anti- body against acrolein (1:500; Cell Sciences, USA). Follow- ing rinses with 0.1 M PBS, sections were treated with a secondary antibody of either rabbit IgG conjugated with Alexa Fluor 594 or mouse IgG conjugated with Alexa Fluor 488 (1:1000, Molecular Probes, OR, USA) for 24 h at 4°C before incubation with the DNA-specific dye bisbenzimide Hoechst (1:10 000) for 2 min. Slides were coverslipped using a mixture of glycerol gelatin (Sigma, St Louis, MO, USA) and water. Confocal and light microscopy analyses Sections were examined, scanned and analysed using Carl Zeiss LSM 5 Pascal confocal microscope (Germany), LM Zeiss Apotome (Germany) and Zeiss Axioplan. During image collection, the photomultiplier settings and expos- ure times for each fluorescent channel were kept constant to allow comparison of protein levels between sections. Quantitative measurement on signal intensity of immunofluorescent sections Digital images of the acrolein and Cox Va-labelled sec- tions obtained from confocal microscopy were processed and analysed using ImageJ software. Consistency in the analyses was ensured by using identical parameters with respect to section thickness (z-stack), areas sampled for analysis, antibody concentrations, duration of incubation times, and microscope configuration. Five identical fields of interest were chosen in the in- ferior region per retina (n = 3). During the image quanti- fication, the described areas were marked by a rectangle drawn from either the tip of the outer segments to outer plexiform layer or along the inner segments. Intensity of fluorescence from each rectangle (one area) was mea- sured and the value was plotted as number of pixels/ field (Cox Va, green; acrolein, red). The values obtained Page 12 of 14 from each animal were averaged and presented in a histogram. RNA isolation and cDNA synthesis Total cellular RNA was extracted from individual retinas and purified using the RNA extraction kit protocol (RNAqueous-Micro kit; Ambion). The purified RNA was quantified on a spectrophotometer (ND-1000; Nanodrop Technologies, Wilmington, DE). The integrity of the sam- ples was assessed using a bioanalyser (2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA). The cDNA was synthesized by reverse transcription using reverse tran- scriptase or first strand cDNA synthesis kit following the manufacturer’s protocols (Superscript III; Invitrogen, Carlsbad, CA). Real time quantitative polymerase chain reaction (RT-qPCR) Quantitative analysis of expression levels for genes Hmox-1 (Mm00516005), C3 (Mm00437838), Fgf-2 (Mm00 433287) and was determined by RT-qPCR using Taqman® probes that were combined with the Gene Expression Master-Mix (Applied Biosystems, Foster City, CA). Step- One Plus qPCR machine was used with the StepOne soft- ware v2.1 (Applied Biosystems) for analysis. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as a refer- ence gene. The variabilities were accounted for by per- forming the Taqman amplification assay in duplicates (individual sample variability) with triplicate biological sam- ples to account for individual animal differences. The fold changes were determined using comparative cycle thresh- old (Ct; delta-delta ct). Statistical analyses Data were analysed using non-parametric tests, Kruskal- Wallis followed by Mann–Whitney U or parametric ANOVA test with Bonferroni corrections. Results are presented as mean ± Standard Error of the Mean (SEM). P value of <0.05 was considered significant. Abbreviations AMD: Age-related macular degeneration; ARVO: Association for research in vision and ophthalmology; ATP: Adenosine triphosphate; BBZ: Bisbenzimide; BRB: Blood-retinal barrier; CCO: Cytochrome c oxidase; Cox Va: Cytochrome oxidase Va; Ct: Cycle threshold; C3: Complement component 3; FC: Fold change; Fgf-2: Fibroblast growth factor 2; FR: Far red; Gapdh: Glyceraldehyde 3-phosphate dehydrogenase; Hmox-1: Heme oxygenase – 1; IHC: Immunohistochemistry; IC: Inferior central; IP: Inferior periphery; ILM: Inner limiting membrane; IS: Inner segments; mRNA: Messenger RNA; NIR: Near infrared; nm: Nanometer; NO: Nitric oxide; NT: Nontreated; OLM: Outer limiting membrane; ON: Optic nerve; ONL: Outer nuclear layer; OS: Outer segments; PUFA: Polyunsaturated fatty acid; qPCR: Quantitative polymerase chain reaction; ROP: Retinopathy of prematurity; ROS: Reactive oxygen species; RP: Retinitis pigmentosa; SC: Superior central; SP: Superior periphery; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling; Tr: Treated. Competing interests The authors declare that they have no competing interests.PDF Image | 670 nm light mitigates oxygen-induced degeneration 2013
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