At DIV10, all three miRNAs exhibited significantly decreased expression relative to DIV3

lavage fluid was harvested and the cell concentration was defined using an automated cell counter. Materials and Methods Ethics Statement All experimental animals were monitored daily by trained animal caretakers. Animals showing signs of distress were euthanized by cervical dislocation. All experiments were conducted according to institutional and national guidelines. The experiments performed in the current project were specifically approved by the Danish Animal Experiments Inspectorate. Following the experiments, animals were euthanized by anaesthetizing using hypnorm/dormicum followed by perfusion, as described below. Liver and Skin 19374401 Samples Liver and skin samples from naive wildtype and Plg2/2 mice were derived from a tissue collection containing samples isolated from mice with an age of eight, 12 and 26 weeks. Histology Paraffin embedded tissues were sectioned, rehydrated and stained using a standard H&E staining protocol. For immunohistochemical detection of fibrin and CD34, the following antibodies were used: rabbit-anti-mouse fibrin diluted 1:2000 and rat-anti CD34 diluted 1:100, followed by incubation with a rabbit-anti-rat antibody diluted 1:100. Chromogen staining was achieved using the EnVision+ system in combination with NovaRED HRP substrate. Stained sections were scanned using a motorized Olympus BX51 microscope with a 20X objective controlled by Visiopharm software or by a NanoZoomer-2.0HT using a 20X or 40X objective. In wound tissue, cell counting was performed in randomly selected areas either 19374401 containing or devoid of fibrin. The average size of these regions of interest was 0.035 mm2. The average thickness of the epidermis was derived from the area of the hyperplastic epidermis divided by the length. The combined area of fibrin rich lesions in the provisional matrix was determined using the staining analysis software VisiomorphDP, which is part of the Visiopharm software package. In the liver, the degree of fibrin deposition was determined on one whole scanned tissue slide from each liver using VisiomorphDP. Likewise, was the area of CD34 positive staining quantitated by use of VisiomorphDP on whole tissue slides scanned at 40X. Animal Breeding Both FVB/n and C57Bl/6 mice were used for wound healing studies. Tissue libraries were derived from FVB/n mice. The thioglycollate induced peritonitis experiments were performed in C57Bl/6 mice. The FVB/n Plg2/2 mice and their littermate controls were generated by breeding heterozygous mice, that had been backcrossed into an FVB/n background for at least 30 generations. C57Bl/6 Plg2/+ mice were likewise backcrossed for at least 30 generations into the C57Bl/6 background. All mice were bred in the SPF facility at University of Copenhagen and during experimentation they were housed individually. Genotyping was performed as previously described and following the experiments all used mice were regenotyped. Ovariectomy Mice of approximately five weeks of age were anaesthetized using hypnorm/dormicum and the ovaries were excised as previously described. Briefly, the ventral skin and muscle tissue were cut with scissors and the ovaries resected and the wound suturated. Similar sham GW 501516 surgeries were performed in both male and female mice. One week following OVX, incisional wounding was performed as described below. At the termination of the experiment, successful OVX was confirmed by isolating and weighing the uteri. 2 Wound Healing in Plasminogen Deficient Mice Statistical Analyses Sta

评论已关闭。