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54 S.Y. Lee et al. / Journal of Photochemistry and Photobiology B: Biology 88 (2007) 51–67 histologic examination with hematoxylin and eosin (H & E) stain, Verhoeff-van Gieson stain, Alcian blue stain (pH 2.5), Schmorl’s stain, and immunohistochemistry. Those taken at two weeks after the final treatment were split vertically into two pieces; one undergoing the same examination as before treatment, and the other being pro- cessed for transmission electromicroscopy (TEM). Any given procedure was performed at the same time by the same technician only after all pre- and post-treatment spec- imens were gathered to avoid any possible biases. 2.6.2. Transmission electromicroscopy The skin specimens were fixed at 4 °C for 3 hours in cold-buffered 2.5% glutaraldehyde in phosphate buffered saline (PBS, pH 7.4) and then post fixed at 4 °C for 1 hour and 30 minutes in 1% osmium tetroxide. After gradual dehydration in an ascending ethanol series, the tissues were transferred into propylene oxide, embedded with an Epon Embedding kit (Ted Pella. Inc. CA. USA) and polymerized at 60 °C for 72 hours in a TD-500 Electron Microscope oven (DOSAKA EM CO. Ltd., Kyoto, Japan). Tissue blocks were trimmed into both thick sections (1lm) which were stained with 1% toluidine blue for light microscopy and thin sections (80 nm) which were double stained with uranyl acetate and lead citrate. The sections were sliced with a Reichert-Jung Ultracut E ultramicrotome (Leica Microsystems, Wetzlar, Germany). All of the thin sections were examined with an H-7600s transmission electron microscope (Hitachi, Tokyo, Japan), 80 kV acceleration voltage. 2.6.3. Immunohistochemical staining Immunohistochemical staining was performed using a peroxide technique. Formalin-fixed paraffin-embedded tis- sue blocks were sectioned to a thickness of 4 lm. Sections were deparaffinized for 5 minutes three times in xylene and rehydrated for 5 minutes per session in serial-graded alcohol (100%, 95%, 80%, 70% alcohol). Antigen retrieval was performed for MMP-1 and TIMP-2. For antigen retrieval, 10 mM citrate buffer (pH 6.0) was heated in a pressure cooker for 10 minutes. After that, the container was cooled for 20 minutes at room temperature. Endoge- nous peroxide activity was blocked by 3% hydrogen per- oxide in methanol for 10 minutes. The slides were washed three times in Tris-buffered saline (TBS, pH 7.6) for 5 minutes and incubated with a blocking solution (normal goat serum) at room temperature for 20 minutes. Table 1 Sequences of the probes and primers for real time RT-PCR The antibodies then used were as follows: an anti-MMP-1 antibody (dilution titer 1:200, Lab Vision, California, USA); an anti-MMP-2 antibody (dilution titer 1:50, Lab Vision): an anti-TIMP-1 antibody (dilution titer 1:100, Lab Vision); and an anti-TIMP-2 antibody (dilution titer 1:50, Lab Vision). The antibodies were incubated for 30 minutes at room temperature and washed three times in TBS for 5 minutes. Subsequently, secondary antibody reaction was achieved with a ChemMate DAKO EnVision Detection Kit (Dako, Denmark) for about 30 minutes at room temperature. After washing with TBS, the samples were stained with 3,30-diaminobenzidine for chromogenic reaction and counter-stained with hematoxylin for 30 seconds. 2.6.4. Real time RT-PCR Total RNA was extracted from skin samples with a Tri- zol kit (Invitrogen Corp., Carlsbad, California). Reverse transcription was performed using 1.5 lg of total RNA in 20lL using a Reverse Transcription Kit (Invitrogen). For real-time PCR assays, a master mix of the following components was prepared, at the indicated final concentra- tions: 2.5 lL each primer (9 lM), 2.5 lL probe (2.5 lM), 2.5 lL water, and 12.5 lL TaqMan PCR 2X master mix- ture (Applied Biosystems, Lincoln, CA). The PCR primers and probes used are listed in Table 1. Five microliters of reverse transcription reaction mixture was added as a PCR template. Relative quantitative real-time PCR was performed using the above reagents using an ABI Prism 7000 Sequence Detection System (Perkin-Elmer Applied Biosystems, Lincoln, CA). The following procedure was used. After initial activation of uracyl-N-glycosylase at 50 °C for 2 minutes, AmpliTaq Gold (Applied Biosystems) was activated at 95 °C for 10 minutes. PCR consisted of 45 amplification cycles (denaturation at 95 °C for 15 seconds, annealing at 60 °C for 1 minute, and extension at 60 °C for 1 minute). During PCR amplification, the amplified prod- uct amount was monitored by continuous measurement of fluorescence. The expression of the genes was normal- ized versus a GAPDH (VIC/MGB probe, primer limited) as follows; the cycle number at which the transcripts of the genes were detectable (threshold cycle, Ct) was normal- ized against the Ct of GAPDH, which is referred to as delta Ct. The expression of the genes relative to a reference was expressed as 2-deltadeltaCt, where deltadeltaCt refers to the difference in the values of deltaCt between the test groups and the reference. Sequence 5 0 -GGAGCAACAAGTGGTGTTCTCCATG-3 0 5 0 -CCCATGTTGTAGCAAACCCTCAAGC-3 0 50-TTCAATGAGGAGACTTGCCTGGTGA-30 5 0 -TCCTCACCGTGTACTGGACTCCAGA-3 0 5 0 -GACCAGTGGTGCGCTGAGCCCTGCC-3 0 IL-1ß TNF-a IL-6 ICAM-1 Connexin 43 Assay ID Hs00174097_m1 Hs00174128_m1 Hs00174131_m1 Hs00164932_m1 Hs00748445_s1PDF Image | LED phototherapy for skin rejuvenation
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