Browsing by Subject "Immunohistochemistry"
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Item Identification of cubilin (p400) as galectin-3 binding protein from the murine utero-placental complex(Texas Tech University, 2000-12) Crider-Pirkle, Sunday Suzanne; Faust, Charles; Weitlauf, Harry M.; Hardy, Daniel; Lee, Vaughan H.; Webster, Daniel R.; Whelly, Sandra M.Galectin-3 is a soluble p-galactoside binding lectin that is present in several cell types within the uteroplacental complex (UPC) of mice. Affinity chromatography with immobilized galectin-3 was used to isolate potential binding partners for the lectin from homogenates of UPC. At least one glycoprotein (Mr 400,000; p400) was isolated that bound galectin-3 in a carbohydrate-dependent manner. Exposure of p400 to glycosidases decreased its apparent size by 10%. Differential migration of p400 in nonreducing and reducing conditions demonstrated that the protein contains intramolecular disulfide bonds. Amino acid sequencing revealed similarity to cubilin, a 400 kDa endocytic receptor. Collectively, the molecular size of p400, its degree of glycosylation, the presence of intramolecular disulfide bonds, and amino acid sequence similarity strongly suggest that p400 is the murine ortholog of cubilin. Immunohistochemistry revealed that cubilin (p400) was present in the yolk sac epithelium from day 8 to term. It was also localized in the perforin-positive granules of uNK cells in metrial gland and decidua basalis. Although cubilin is best known as the receptor for intrinsic factor-vitamin B12 in the ileum, it may also act as an endocytic receptor in the kidney and yolk sac where it presumably mediates transcytosis of multiple ligands. The localization of cubilin to uNK cells is the first demonstration of the protein in an immune or non-epithelial cell type. However, the questions of whether cubilin actually interacts with galectin-3 in vivo, and what role cubilin plays, cannot be answered from our results. Because both galectin-3 and cubilin are present in uNK cells, one intriguing hypothesis is that they interact to modulate the immune function of uNK cells, and thus, that they are a part of a mechanism for protecting the fetus from immune rejection.Item An immunohistochemical analysis of regenerating cellular material in two distinct models of skeletal muscle injury(2011-08) Sarathy, Apurva; Farrar, Roger P.; Suggs, Laura J.Tourniquet mediated Ischemia Reperfusion (I/R) injury causes damage to skeletal muscle, often resulting in prolonged functional impairment. The current study utilizes immunohistochemistry (IHC) to determine whether the controlled release of the anabolic factor, insulin-like growth factor-I (IGF-I), from the biodegradable PEGylated fibrin gel matrix can facilitate the recovery of skeletal muscle from I/R. Treatment groups following a 2-hour tourniquet applied to the limb of 6-9 month rats, included intramuscular injections of saline, PEGylated fibrin gel (PEG-Fib) only and IGF-I conjugated to PEGylated fibrin gel (PEG-Fib-IGF). Expression of the myogenic regulatory factors MyoD and myogenin detected via IHC in the PEG-Fib-IGF group was significantly lower compared to the saline group, showing a 1.4±0.8% nuclear co-localization for MyoD and a 2.0±0.8% nuclear co-localization for myogenin at 14 days of recovery. The saline group showed higher values, 31.4±4.4% and 44.1±7.3% for MyoD and myogenin nuclear co-localization respectively. A significantly greater percentage, 88.8±3.7% of Desmin positive myofibers was seen at 14 days of recovery, while a lower percentage of fibers expressing neonatal myosin, 7.7±2.7% was seen in the PEG-Fib-IGF group compared to the saline treatment group. These results indicate that IGF-I delivered intramuscularly via PEGylated fibrin gel, functions therapeutically in skeletal muscle recovery, from I/R mediated damage. In a separate injury model that deals with volumetric muscle loss, IHC analyses were performed to test the efficacy of a novel tissue engineering strategy utilizing extracellular matrix (ECM) as a scaffold. In this model, also called the defect model, a 1.0 X 1.0 cm piece of the lateral gastrocnemius was removed and replaced with a muscle-derived ECM. The constructs were then seeded with bone marrow derived cells (BMSCs), adipose derived stem cells (ADSCs) or the peroneal nerve was relocated to the area of the ECM implant. 42 days post recovery IHC analysis was performed on the ECM implants. The quantification of desmin-positive regenerating myofibers bearing centrally located nuclei, showed significantly greater values in the top, middle and bottom region of the ECM implants that received peroneal nerve relocation, when compared to the experimental group that received the ECM implant alone. Blood vessel density increases were seen within the middle region of the ECM implant groups that received BMSC+Nerve treatment and the bottom region of the ECM implant groups that received ADSC+Nerve treatment. Thus, these results corroborate the therapeutic effect of peroneal nerve relocation, which stimulated an increase in myofiber regeneration and vascular maintenance within the construct.