Secondary antibody in electron microscopy

Secondary antibody in electron microscopy


In electron microscopy, to detect specific macromolecule or structure such as spindle pole body (SPB), the frequently used procedure is to the couple secondary antibody with

  1. Alexa 568
  2. Cy5
  3. Gold particle
  4. Osmium tetraoxide

Rephrasing the Question:

What do we use for indirect labelling of secondary antibody in electron microscopy?


Gold particles coupled to a secondary antibody (option 3).


Each of these is used in microscopy, but only one is used for indirect labelling (using an antibody) in electron microscopy.

  1. Alexa Fluor dyes are fluorescent compounds used as cell and tissue labels in fluorescence microscopy. Alexa Fluor 568 has an excitation peak at 579 nm and an emission peak at 603 nm.
  2. Cy5 is a type of cyanine dye. Cyanine dyes are used in light microscopy and related applications, to label proteins, antibodies, peptides, and nucleic acids. It is especially used in a variety of fluorescence detection techniques (e.g., flow cytometry, immunofluorescence microscopy, and microplate assays). It is far-red fluorescent with excitation at 633 nm.
  3. Immunogold labelling is a technique used in electron microscopy. Colloidal gold particles are attached to secondary antibodies which are in turn attached to primary antibodies designed to bind a specific antigen or other cell component. Gold is used for its high electron density, so it provides high contrast.
  4. Osmium tetraoxide (OsO4) is also used in electron microscopy, but it is used for direct visualization, as a lipid stain. It embeds a heavy metal directly into cell membranes by binding to the phospholipid heads, creating contrast with the cytoplasm.
Secondary antibody in electron microscopy


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