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Multitarget Immunohistochemistry for Confocal and Super-resolution Imaging of Plant Cell Wall Polysaccharides.

ABSTRACT: 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.

SUBMITTER: Haas KT

PROVIDER: S-EPMC7842508 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Like the brain, the human inner ear was long thought to be devoid of immune activity. Only the endolymphatic sac (ES) was known to be endowed with white blood cells that could process antigens and serve as an immunologic defense organ for the entire inner ear. Unexpectedly, the cochlear and vestibular organs, including the eighth cranial nerve, were recently shown to contain macrophages whose functions and implication in ear disease are somewhat undefined. Here, we review recent inner ear findings in man and extend the analyses to the vestibular nerve using super-resolution structured illumination microscopy (SR-SIM). Materials and Methods: Human ESs and cochleae were collected during surgery to treat patients with vestibular schwannoma and life-threatening petro-clival meningioma compressing the brainstem. The ESs and cochleae were placed in fixative, decalcified, and rapidly frozen and cryostat sectioned. Antibodies against ionized calcium-binding adaptor molecule 1-expressing cells (IBA1 cells), laminin β2 and type IV collagen TUJ1, cytokine fractalkine (CX3CL1), toll-like receptor 4 (TLR4), CD68, CD11b, CD4, CD8, the major histocompatibility complex type II (MHCII), and the microglial marker TEME119 were used. Results: IBA1-positive cells were present in the ESs, the cochlea, central and peripheral axons of the cochlear nerve, and the vestibular nerve trunk. IBA1 cells were found in the cochlear lateral wall, spiral limbus, and spiral ganglion. Notable variants of IBA1 cells adhered to neurons with "synapse-like" specializations and cytoplasmic projections. Slender IBA1 cells occasionally protracted into the basal lamina of the Schwann cells and had intimate contact with surrounding axons. Discussion: The human eighth nerve may be under the control of a well-developed macrophage cell system. A small number of CD4+ and CD8+ cells were found in the ES and occasionally in the cochlea, mostly located in the peripheral region of Rosenthal's canal. A neuro-immunologic axis may exist in the human inner ear that could play a role in the protection of the auditory nerve. The implication of the macrophage system during disease, surgical interventions, and cell-based transplantation should be further explored.

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Multitarget Immunohistochemistry for Confocal and Super-resolution Imaging of Plant Cell Wall Polysaccharides.

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