Kearnscortez2654

Our live-imaging approach is also applicable to C. elegans hermaphrodites and should be expandable to other fluorescently labelled nematodes or other fully transparent small model organisms.Oxygenic photogranules (OPGs) are dense, three-dimensional aggregates containing a syntrophic, light-driven microbial community. Their temporal and spatial development interests microbial ecologists working at the bioprocess engineering interface, as this knowledge can be used to optimize biotechnological applications, such as wastewater treatment and biomass valorization. The method presented here enables the high-throughput quantification of photogranulation. OPGs are produced from a loose sludge-like microbial matrix in hydrostatic batch cultures exposed to light. This matrix transforms into a consolidated, roughly spherical aggregate over time. Photogranulation is quantified by time-lapse imaging coupled to automated image analysis. This allows studying the development of many OPGs simultaneously and in a fully automated way to systematically test what factors drive photogranulation. The protocol can also be used to quantify other types of (a)biotic aggregation.The plant cell wall (PCW) is a pecto-cellulosic extracellular matrix that envelopes the plant cell. By integrating extra-and intra-cellular cues, PCW mediates a plethora of essential physiological functions. Notably, it permits controlled and oriented tissue growth by tuning its local mechano-chemical properties. To refine our knowledge of these essential properties of PCW, we need an appropriate tool for the accurate observation of the native (in muro) structure of the cell wall components. The label-free techniques, such as AFM, EM, FTIR, and Raman microscopy, are used; however, they either do not have the chemical or spatial resolution. Immunolabeling with electron microscopy allows observation of the cell wall nanostructure, however, it is mostly limited to single and, less frequently, multiple labeling. Immunohistochemistry (IHC) is a versatile tool to analyze the distribution and localization of multiple biomolecules in the tissue. The subcellular resolution of chemical changes in the cell wall component can be observed with standard diffraction-limited optical microscopy. Furthermore, novel chemical imaging tools such as multicolor 3D dSTORM (Three-dimensional, direct Stochastic Optical Reconstruction Microscopy) nanoscopy makes it possible to resolve the native structure of the cell wall polymers with nanometer precision and in three dimensions. Here we present a protocol for preparing multi-target immunostaining of the PCW components taking as example Arabidopsis thaliana, Star fruit (Averrhoa carambola), and Maize thin tissue sections. This protocol is compatible with the standard confocal microscope, dSTORM nanoscope, and can also be implemented for other optical nanoscopy such as STED (Stimulated Emission Depletion Microscopy). The protocol can be adapted for any other subcellular compartments, plasma membrane, cytoplasmic, and intracellular organelles.Flavonols are a subclass of flavonoids of the group of plant secondary metabolites. In planta, flavonols play various functions such as antioxidant and natural regulator of auxin polar transport. Many lines of evidence have shown that flavonols also contribute to human health in anti-oxidation, anti-inflammation, and even prevention some types of cancer. Several methods have been utilized to measure flavonols such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and diphenylboric acid-2-aminoethyl ester (DPBA) staining. While HPLC or LC-MS can quantitatively determine the level of flavonols, DPBA staining can provide an in-situ view of flavonols accumulation in the plants. In this protocol, a detailed procedure for staining the flavonols in Arabidopsis root tips is described. Five-day-old Arabidopsis seedlings are soaked in a solution containing DPBA and latterly the flavonols (kaempferol and quercetin) can be observed under a confocal microscope.Bacteria are surrounded by a protective peptidoglycan cell wall. Provided that this structure and the enzymes involved are the preferred target for our most successful antibiotics, determining its structural and chemical complexity is of the highest interest. Traditionally, high-performance liquid chromatography (HPLC) analyses have been performed, but these methods are very time consuming in terms of sample preparation and chromatographic separation. Here we describe an optimized method for preparation of Gram-negative bacteria peptidoglycan and its subsequent analysis by ultra-performance liquid chromatography (UPLC). The use of UPLC in peptidoglycan analyses provides a dramatic reduction of the sample volume and hands-on time required and, furthermore, permits in-line mass spectrometry (MS) of the UPLC resolved muropeptides, thus facilitating their identification. Selleckchem Darovasertib This method improves our capability to perform high throughput analysis to better understand the cell-wall biology.Growing evidences suggest that peritubular capillaries pericytes are the main source of scar-forming myofibroblasts during chronic kidney disease (CKD), as well as early phases of acute kidney injury (AKI). In a swine model of sepsis and I/R (Ischemia Reperfusion) injury-induced AKI we demonstrated that renal pericytes are able to transdifferentiate toward α-SMA+ myofibroblasts leading to interstitial fibrosis. Even if precise pericytes identification requires transmission electron microscopy and the co-immunostaining of several markers (i.e., Gli, NG2 chondroitin sulphate proteoglycan, CD146, desmin or CD73) and emerging new markers (CD248 or TEM1, endosialin), previous studies suggested that PDGFR-β could be used as marker for renal pericytes characterization. Recently, double immunofluorescence staining of PDGFR-β and α-SMA was performed to identify the damage activated pericytes (PDGFR-β+/α-SMA+ cells) in the early phase of fibrosis development. Our data highlighted the crucial role of renal pericytes in the physiopathology of sepsis and I/R associated AKI. In this protocol, we describe the procedure for double immunofluorescence staining of PDGFR-β and α-SMA in swine Formalin-Fixed Paraffin-Embedded (FFPE) kidney biopsies and the method for image analysis and quantification.