1) Immunostaining. Using anti-zip8 for cell staining, what does it stain? 2) how
ID: 62781 • Letter: 1
Question
1) Immunostaining. Using anti-zip8 for cell staining, what does it stain?
2) how do we visualize and analyize results while performing cell staining with DAPI and anti-zip8? What do the results of staining imply?
3) Immunostaining (what is primary antibody? What is secondary antibody? Why we need a secondary antibody?)
4) H&E stain of mouse liver. what is the morphology of normal liver? what is the morphology of abnormal liver? what is the pathology?
5) Immunostaining (what is primary antibody? What is secondary antibody? Why we need a secondary antibody?)
Explanation / Answer
1)
anti-zip 8 identifies zip 8 glycosylated protein (a member of a family of divalent ion transporters for iron and zinc) located at the plasma membrane (mostly), early endosome and mitochondria and thus anti-zip stains plasma membrane, early endosome and mitochondria.
2.
DAPI is a popular nuclear and chromosome counterstain used in immunofluorescenec to provide the information whether the protein in study is present in cytoplasmic, nuclear or plasma membrane. DAPI emits blue fluorescence upon binding to AT regions of DNA and anti-zip8 with different fluorescence (say red) is used to find cellular localization of zip8 protein. If we observe blue staining in nucleus and red fluorescence all over except nuclear region then we conclude zip8 protein is cytoplasmic and plasma membrane bound. To make more specific we take advantage of markers specific to organelles to find the precise location of zip8 protein within cell. For confirming the cell surface presence, we need not to permeabilize the cell and if two colours are visible (one nuclear and other at membrane) then presence of zip8 protein at surface is confirmed. If zip8 protein shows nuclear presence then the colour will be blend of both fluorescences.
Taking into account the cellular localization of zip8 we expect blue staining of nucleus and red fluorescence outside nucleus or more staining at cell surface.
3.
A primary antibody is an immunoglobulin that specifically binds to a particular protein or other biomolecule of research interest for the purpose of purifying or detecting and measuring it. Primary antibodies are developed as polyclonal or monoclonal antibodies using mouse, rat, rabbit, goat, and other animal species as hosts.
Secondary antibodies are used for the indirect detection of target antigens. They may be conjugated or unconjugated and can be used for the fluorescent, colorimetric, and chemiluminescent detection of primary antibodies in a wide range of applications, such as cell imaging, flow cytometry and western blotting.
4.
H & E staining of Normal Liver: Portal areas, containing elements of the hepatic triad, that is one or more small branches of the portal vein, a branch of the hepatic artery, and a small bile duct, along with lymphatic vessels and a very small amount of connective tissue, could be identified. The presence of portal areas along with central venules provided evidence of a lobular structure to the liver. Liver cells are arranged in plates or cords, and are seen radiating from the regions of central venules and extending to the portal areas. The plates or cords of cells are separated by sinusoidal capillaries. Tissue processed with H&E (or with the fluorescent DAPI stain, see below) reveals distinctly stained nuclei of varied shapes. Most common are cells having one, or sometimes two, large round to slightly oval nuclei. Also detected in the H&E stained, or DAPI labeled, sections are other cells with smaller and darkly staining oval or oblong nuclei.
H & E staining of abnormal Liver: Severe hepatocellular injury with widespread necrosis and inflammation and remarkable nuclear enlargement with prominent nucleoli and vacuolization. Focal proliferation of bile ducts, including elongation and focal proliferation of small oval-like cells in the portal tract, accompanied by acute cholangitis. Fibrosis in relation to chronic inflammation in portal tracts around the bile ducts. Scattered macrophages, some forming multinucleated cells, and focal steatosis also detected. Extremely distorted ducts lined by multiple layers of cells with high nuclear-cytoplasmic ratio, hyperchromasia, and dispolarity, consistent with displasia and probable carcinomatous transformation. Marked complexity and irregularity of the tubular structures and significant pleomorphism of the epithelium, with the loss of usual basal nuclear polarity, anisonucleosis, and occasional mitoses.
Above patholog is as a result of copper accumulation in liver of patients suffering from Wilson Disease.
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