Probes for GFAP

The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to simultaneously assess transcription and chromatin accessibility in EGCs undergoing spontaneous neurogenesis in culture, as well as small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. A subset of in vivo EGCs, highly enriched within the myenteric ganglia and that persist into adulthood, have a gene expression program and chromatin state consistent with neurogenic potential. These results clarify the mechanisms underlying EGC potential for neuronal fate transition.

We identified 2 types of cells, with positive immunohistochemical reaction to EGFR in pituitary tumors with negative reaction to EGFR. In some adenomas, we identified, at the periphery of the tumor, an important accumulation of tumor associated macrophages. In these cells, we identified a strong expression of the receptor EGFR. We identified a strong expression of EGFR in folliculostellate cells with a homogenous or granular cytoplasmatic pattern.Both adherent and gap junctions are between folliculostellate cells and between folliculostellate cells and endocrine cells. These cells are also positive for GFAP (glial fibrillary acidic protein), suggesting that this cell type may represent an astrocyte – or microglia – like cell type. Our observations support older findings, notably the increased activity of folliculostellate cells under pathological conditions, their phagocytic activity and their capacity to secrete angiogenic growth factors, suggesting that folliculostellate cells may be involved in basement membrane remodeling, tumoral neoangiogenesis and tumoral expansion.

Angiotensin (Ang) II signalling in the hypothalamic paraventricular nucleus (PVN) via angiotensin type-1a receptors (AT1R) regulates vasopressin release and sympathetic nerve activity - two effectors of blood pressure regulation. We determined the cellular expression and function of AT1R in the PVN of a rodent model of polycystic kidney disease (PKD), the Lewis Polycystic Kidney (LPK) rat, to evaluate its contribution to blood pressure regulation and augmented vasopressin release in PKD.PVN AT1R gene expression was quantified with fluorescent in-situ hybridisation in LPK and control rats. PVN AT1R function was assessed with pharmacology under urethane anaesthesia in LPK and control rats instrumented to record arterial pressure and sympathetic nerve activity.AT1R gene expression was upregulated in the PVN, particularly in CRH neurons, of LPK versus control rats. PVN microinjection of Ang II produced larger increases in systolic blood pressure in LPK versus control rats (36±5 vs. 17±2 mmHg; P<0.01). Unexpectedly, Ang II produced regionally heterogeneous sympathoinhibition (renal: -33%; splanchnic: -12%; lumbar no change) in LPK and no change in controls. PVN pre-treatment with losartan, a competitive AT1R antagonist, blocked the Ang II-mediated renal sympathoinhibition and attenuated the pressor response observed in LPK rats. The Ang II pressor effect was also blocked by systemic OPC-21268, a competitive V1A receptor antagonist, but unaffected by hexamethonium, a sympathetic ganglionic blocker.Collectively, our data suggest that upregulated AT1R expression in PVN sensitises neuroendocrine release of vasopressin in the LPK, identifying a central mechanism for the elevated vasopressin levels present in PKD.The Author(s).

Abstract Purpose: Simultaneous dual labeling to visualize specific RNA and protein content within the same formalin-fixed paraffin embedded (FFPE) section can be technically challenging and usually impossible, because of variables such as tissue fixation time and pretreatment methods to access the target RNA or protein. Within a specific experiment, ocular tissue sections can be a precious commodity. Thus, the ability to easily and consistently detect and localize cell-specific expression of RNA and protein within a single slide would be advantageous. In this study, we describe a simplified and reliable method for combined in situ hybridization (ISH) and immunohistochemistry (IHC) for detection of mRNA and protein, respectively, within the same FFPE ocular tissue. Methods: Whole mouse eyes were prepared for 5 micron FFPE sections after fixation for 3, 24, 48 or 72 h. Customized probes from Advanced Cell Diagnostics to detect mRNA for vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), and hypoxia-inducible factor 2-alpha (HIF-2α) were used for ISH. Various parameters were tested using the novel RNAscope method for ISH and optimized for compatibility with subsequent IHC for glial fibrillary acidic protein (GFAP) or GS-lectin within the same tissue section. Dual fluorescent visualization of Fast Red ISH and Alexa Fluor 488 IHC signal was observed with confocal microscopy. Results: A fixation time of 72 h was found to be optimal for ISH and subsequent IHC. The RNAscope probes for VEGF, HIF-1α, and HIF-2α mRNA all gave a strong Fast Red signal with both 48 h and 72 h fixed tissue, but the optimal IHC signal for either GFAP or GS-lectin within a retinal tissue section after ISH processing was observed with 72 h fixation. A pretreatment boiling time of 15 min and a dilution factor of 1:15 for the pretreatment protease solution were found to be optimal and necessary for successful ISH visualization with 72 h FFPE ocular tissue. Conclusions: The protocol presented here provides a simple and reliable method to simultaneously detect mRNA and protein within the same paraffin-embedded ocular tissue section. The procedure, after preparation of FFPE sections, can be performed over a 2-day or 4-day period. We provide an optimization strategy that may be adapted for any RNAscope probe set and antibody for determining retinal or ocular cell-specific patterns of expression.