Alkaline phosphatase

8-OxodG was measured by high-performance liquid chromatography with electrochemical detection (HPLC/EC) as described previously [33 (link)]. Briefly, DNA was resuspended in Tris-EDTA, incubated with RNases A and T1 at 37 °C for 1 h, and precipitated again with ethanol. Enzymatic digestion was then performed at 37 °C using nuclease P1 (Boehringer Mannheim) for 2 h and alkaline phosphatase (Boehringer Mannheim, Mannheim, Germany) for 1 h. Enzymes were precipitated by addition of CHCl3, and the upper liquid layer stored at −80 °C under N2 for subsequent analysis 8-oxodG. The DNA hydrolysate was analyzed by high-performance liquid chromatography with electrochemical detection (Coulochem I; ESA, Inc., Chelmsford, MA, USA) using a C18 250 × 46 mm 5-μm Uptishere column (Interchim) equipped with a C18 guard column. The eluent was 50 mm ammonium acetate (pH 5.5), containing 9% methanol, at a flow rate of 0.7 mL/min. The potentials applied were 150 and 400 mV for E1 and E2, respectively. The retention time of 8-oxodG was ~23 min. Deoxyguanosine was measured in the same run of corresponding 8-oxodG with a UV detector (model SPD-2A; Shimadzu, Kyoto, Japan) at 256 nm; the retention time was ~17 min.

Southern blot is a hybridization-based technique that can effectively identify transgenic insert/copy numbers [68 (link)]. The total genomic DNA (10 μg) was extracted according to the manufacturer's instructions (QIAGEN) and digested by the BbsI restriction enzyme (NEB). Genomic DNA fragments were transferred to a membrane, and incubating specific probe targeting mfat-1 gene amplificated with digoxigenin (DIG)-dUTP (Roche) at 65℃ over 16 h in a hybridization oven (primers used for probe amplification, forward: 5´-GGACCTGGTGAAGAGCATCCG-3´; reverse: 5´-GCCGTCGCAGAAGCCAAAC-3´). Then the membrane was incubated with an anti-DIG antibody conjugated with alkaline phosphatase (AP) (Roche) for 1 h at RT. The mfat-1 gene was detected by the CDP star (Roche), and the corresponding image was captured using a ChemiDoc Touch Imaging System (Bio-Rad).

The contraction abilities of the FBs and DP cells were assessed on the basis of the expression of the specific cytoskeleton proteins vimentin (Abcam, USA) and α-smooth muscle actin (α-SMA; (Abcam, USA) involved in collagen gel contraction.
Proliferating cells were identified by positive staining for Ki67 (Abcam, USA). The presence of specific stem cell markers secreted by the DP cells were assessed by the expression of versican (Abcam, USA) and staining with alkaline phosphatase (Roche Diagnostics, Schweiz), respectively.
The immunohistochemistry and alkaline phosphatase staining was carried out according to standard protocols. The primary antibodies were diluted in the following concentrations: vimentin, Ki67, versican, 1:200; α-SMA, 1:50.
To detect the primary antibodies, secondary antibodies were used conjugated with Alexa-488 or Alexa-546 (dilution 1:200). The nuclei were counterstained with 0.001% DAPI (Santa Cruz, Texas, USA). The stained dermal equivalents were examined using a fluorescence microscope for the purposes of the immunohistochemistry. The immunohistochemical analysis of the prepared dermal equivalents was carried out on days 1, 7, and 14 of cultivation.

Mouse eyes were fixed overnight in 4% PFA, cryoprotected with 30% sucrose, and embedded in OCT. Twenty‐micrometer cryosections were treated with 5 μg/ml proteinase K for 15 min. After washes with 2 mg/ml glycine and post‐fixation with 4% PFA/0.2% glutaraldehyde, sections were prehybridized with 50% formamide, 5× sodium saline citrate buffer (SSC) and citric acid to pH 6.1% sodium dodecyl sulfate (SDS), 500 μg/ml yeast tRNA, and 50 μg/ml heparin. The miRCURY detection miR‐181a and miR‐181b locked nucleic acid probes (Exiqon) were used at a final concentration of 30 nM. Probe hybridization was performed overnight at 42°C. Hybridized sections were washed with 50% formamide, 2 × SSC at the hybridization temperature. Sections were blocked for 1 h in MABT (100 mM maleic acid, 150 mM NaCl, and 0.1% Tween‐20) containing 1% blocking reagent (Roche) and 10% sheep serum and incubated with alkaline phosphatase (AP)‐labeled anti‐digoxigenin antibody (1:2,000; Roche) in MABT/1% blocking reagent overnight at 4°C. Sections were stained with NBT‐BCIP (Roche) and photographed under a Leica DM5000 microscope.

Restriction enzymes, alkaline phosphatase, and ampicillin were obtained from Roche. The DNA Blunt T/A ligase and Taq-Q5 High-Fidelity DNA polymerase were from New England Biolabs. Plasmid DNA preparation and DNA extraction from agarose gel were carried out using PureYield™ Plasmid miniprep system (Promega) and QIAEX II Gel Extraction Kit (Qiagen), respectively.
Ingredients for bacterial growth were from Difco. Bromocresol green was from Fluka; L-Glutamic acid, L-Glutamine, D,L-Norvaline, sodium acetate, sodium chloride, Triton X-100, GABA, and other reagents, if not otherwise stated, were from Sigma–Aldrich. Super gradient grade methanol for HPLC was from VWR. 2-o-phthalaldehyde (OPA) was from Agilent Technologies. Oligonucleotide synthesis and DNA sequencing services were by MWG Biotech.

All ELISAs were performed in principle as described by Schweiger et al., (2012) [20 (link)] and the antigens were coated according to previous titration studies [20 (link)]. In this study, an anti-human-immunoglobulin G (IgG; polyclonal rabbit anti-Human-IgG, specific for the Fc part of the heavy chain; DAKO, Glostrup, Denmark) conjugated to alkaline phosphatase (Roche Applied Science, Rotkreuz, Switzerland) was used with a dilution of 1:500. Positive standard sera of human patients with confirmed AE or CE and negative standard sera of blood donors were included in all test runs and ELISA plates (5 sera per plate).