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Expansion microscopy (ExM) is a microscopic imaging approach that can achieve super-resolution visualization of fluorescently labeled biological samples using conventional fluorescence microscopy. The method is based on embedding of a fluorescently labeled biological sample in a hydrogel matrix followed by the physical expansion of the specimen, which is then viewed using a conventional fluorescent microscope. Variations of the method can be used to visualize endogenously expressed fluorescent proteins, such as GFP, fluorescently tagged antibodies, nucleic acids, or other fluorescently tagged molecules. A significant challenge of the method is that the physical expansion of the specimen produces a concommitant reduction in fluorescence intensity, which can make imaging difficult. We describe an approach for amplifying fluorescence signal following expansion of immunolabeled tissue sections by applying fluorescently labeled Fab fragment secondary antibodies to intensify fluorescent signal and enhance detection of labeling using conventional fluorescent microscopy. A method to increase immunofluorescence signal intensity of Expansion Microscopy specimens is described. Method utilizes commercially available reagents. Enhances ability to acquire useful images in expanded tissue samples.

Mild traumatic brain injury (mTBI) diagnoses have increased due to aggressive sports and blast-related injuries, but the cellular mechanisms and pathology underlying mTBI are not completely understood. Previous reports indicate that Nociceptin Orphanin/FQ (N/OFQ), an endogenous neuropeptide, contributes to post-injury ischemia following mechanical brain injury, yet its specific role in cerebral hypoxia, vestibulomotor function and injury marker expression following blast-induced TBI is not known. This study is the first to identify a direct association of N/OFQ and its N/OFQ peptide (NOP) receptor with TBI-induced changes following a single 80psi head blast exposure in male rats. N/OFQ and NOP receptor expression increased in brain tissue and plasma following TBI, concurrent with vestibular dysfunction but preceding hypoxia and appearance of injury markers compared to sham rats. A single post-blast treatment with the NOP receptor antagonist, SB-612111, transiently improved acute vestibulomotor performance. It also prevented increases in markers of TBI-induced hypoxia, pro-apoptotic proteins and injury seen 8-10days post-blast. This study reveals an apparent role for the N/OFQ-NOP receptor system in blast TBI and suggests potential therapeutic utility of NOP receptor antagonists for mTBI.

During wound healing, fibroblasts transition from quiescence to a migratory state, then to a contractile myofibroblast state associated with wound closure. We found that the myofibroblast phenotype, characterized by the expression of high levels of contractile proteins, suppresses the expression of the pro-migratory gene, MMP-2. Fibroblasts cultured in a 3-D collagen lattice and allowed to develop tension showed increased contractile protein expression and decreased MMP-2 levels in comparison to a stress-released lattice. In 2-D cultures, factors that promote fibroblast contractility, including serum or TGF-β, down-regulated MMP-2. Pharmacologically inducing F-actin disassembly or reduced contractility increased MMP-2 expression, while conditions that promote F-actin assembly suppressed MMP-2 expression. In all cases, changes in MMP-2 levels were inversely related to changes in the contractile marker, smooth muscle α-actin. To determine if the mechanisms involved in contractile protein gene expression play a direct role in MMP-2 regulation, we used RNAi-mediated knock-down of the myocardin-like factors, MRTF-A and MRTF-B, which induced the down-regulation of contractile protein genes by fibroblasts under both serum-containing and serum-free conditions. In the presence of serum or TGF-β, MRTF-A/B knock-down resulted in the up-regulation of MMP-2; serum-free conditions prevented this increased expression. Together, these results indicate that, while MMP-2 expression is suppressed by F-actin formation, its up-regulation is not simply a consequence of contractile protein down-regulation.

Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.

Amino acid analysis using high-performance liquid chromatography demonstrated high levels of the excitatory amino acids, aspartate and glutamate, in the retinas of congenitally blind chicks at the time of photoreceptor degeneration. Concentrations of aspartate were about 2 times higher in blind chicks than in retinas of age-matched sighted chicks that were carriers for the genetic defect. Glutamate levels were similar in blind chicks and carriers at 1 day of age, but doubled and tripled sighted chick values at 1 week and 2 weeks of age in blind chick retinas. Light microscopic immunocytochemistry using antibodies that recognize aspartate and glutamate revealed increased levels of these two amino acids specifically in the photoreceptor layer of blind chicks. This report is the first to demonstrate high endogenous levels of excitatory amino acids associated with a hereditary degeneration of photoreceptor cells.

Immunoreactivities of two monoclonal antibodies (MAbs) that recognize cone photopigments were tested in the retinas of congenitally blind retinal degenerate ( rd ) chicks and compared to normally sighted carrier chicks, heterozygous for the mutation. MAb OS-2 had been previously determined to label rod and most cone outer segment membranes in normal chick retinas and is believed to bind to an epitope that is common to several photopigments in chickens. MAb COS-1 labels specifically middle-to-long-wavelength-sensitive cone photopigments in a number of vertebrate species. In rd chicks MAb OS-2 labeled the same number of rod outer segments at the same densities as carrier chicks. However, cone outer segments were less frequently and significantly less heavily labeled with this MAb at all ages tested (1 day, 1 week and 2 weeks post hatching). MAb COS-1 labeled the same number of cone outer segments in both rd and carrier retinas at 1 day of age, however, those outer segments that were labeled in rd specimens had significantly fewer gold particles on them. At both 1 week and 2 weeks of age, rd chick retinas had a significant reduction in numbers of cone outer segments labeled by COS-1. These findings support the hypothesis that the cone photopigment protein is abnormal in the rd chick model of hereditary blindness and retinal degeneration.