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ACCEPTED MANUSCRIPT Alexa-Fluor-488 (S32453; Thermo Fisher Scientific). Fluorescence was visualized using a + laser-scanning A1 confocal microscope (Nikon, Tokyo, Japan) and captured with NIS- Element AR software (Nikon). 2.9 Quantitative real-time PCR (qPCR) Retinas were collected and stored in RNA stabilizer (RNAlater; Thermo Fisher Scientific) overnight at 4°C. RNA extraction was performed described previously (Rutar et al., 2011a), using a combination of TRIzol (Thermo Fisher Scientific) and an RNAqueous Total RNA Isolation kit (Thermo Fisher Scientific) following the manufacturer’s instructions. cDNA was synthesised from extracted RNA using a Tetro cDNA Synthesis Kit (Bioline, London, UK), according to the manufacturer’s protocol. Gene expression was determined by real-time quantitative PCR (qPCR), using Taqman hydrolysis probes (Table 1; Thermo Fisher Scientific) and Taqman Gene Expression Master Mix (Thermo Fisher Scientific), which were applied according to the manufacturer’s instructions. qPCR reactions were run in duplicate using a QuantStudio Flex 12K instrument (Thermo Fisher Scientific). Data analysis was performed using the comparative cycle threshold method (∆∆Ct), which was normalised to the expression of the Gapdh reference gene. 2.10 In situ hybridization To localise Ccl2 mRNA expression in retinas, Ccl2 was cloned from PCR products (550-bp amplicon) using cDNA synthesis from retinal RNA (as described above). A digoxigenin (DIG)-labelled riboprobe for Ccl2 mRNA was synthesised according to our previous publication (Rutar et al., 2011b). In situ hybridisation was used on retinal cryosections as described previously (Cornish et al., 2005). Briefly, the Ccl2 riboprobe was hybridized overnight at 55°C and then washed in a series of saline sodium citrate solutions (pH 7.4) at 60°C. The bound probe was visualised using NBT/BCIP. 10 ACCEPTED MANUSCRIPTPDF Image | Photobiomodulation with 670 nm light ameliorates retinal
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