Immunofluorescence

Immunofluorescence (IF) is an umbrella term for several light microscopy techniques such as epi-fluorescence microscopy, confocal microscopy or various super-resolution microscopy methods. In IF, target structures are visualized using fluorescently labelled antibodies or Nanobodies that bind specifically to the target antigen.

Two methods of antigen detection can be distinguished:

  • Direct labeling:
    A primary, labeled antibody binds directly to the antigen.
  • Indirect labeling:
    A primary, unlabeled antibody binds to the target antigen. The primary antibody is detected by a secondary, labeled antibody.

Immunocytochemistry

Immunocytochemistry (ICC) can be used to visualize specific antigens of structurally intact cells with antibodies or antibody fragments. For staining of intracellular targets, cells are fixed and permeabilized. Among other applications, ICC allows the monitoring of protein expression levels and protein localization.

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Immunofluorescence of Spot-tagged proteins

Spot-tagged proteins can be detected by immunofluorescence using various microscopy techniques, including epi-fluorescence, confocal, and super resolution microscopy. ChromoTek’s Spot-Label® offers a choice of different fluorophores for detection. Spot-Label is the first Nanobody used in super resolution microscopy for the detection of a peptide tag. Because of its small size, Spot-Label has a minimal epitope-to-label displacement, which leads to better resolution. In addition, the small size allows excellent tissue penetration.

The Spot-Tag is an inert 12 amino acid peptide-tag (PDRVRAVSHWSS), which is specifically bound by the highly stable and robust anti-Spot-Nanobody used in Spot-Label.

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Immunohistochemistry

In immunohistochemistry (IHC), tissue sections can be stained with fluorescent probes. The cell morphology and the tissue architecture are preserved by various fixation methods.

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Tissue clearing

Tissue clearing techniques are used to make tissues, organs or even whole animals transparent. The cleared samples can be analyzed by light microscopy (e.g. epi fluorescence, confocal, two-photon, and light sheet fluorescence microscopy). There are several approaches to achieve tissue clearing as reviewed in Richardson and Lichtman (2017):

  • Solvent-based clearing (Spalteholz, 3Disco, iDisco, etc.)
  • Simple immersion (Sucrose, ClearTa, etc.)
  • Hyperhydration (Scale U2, CUBIC, etc.)
  • Hydrogel embedding (CLARITY, PARS, etc.)

Some of these techniques are compatible with immunostaining. Using IgG antibodies, very long incubation times are required for sufficient tissue penetration. Due to their small size, fluorescently labelled Nanobodies, i.e. Nano-Boosters, Nano-Labels, and Nano-Secondaries, offer the advantage of considerably better tissue penetration and thus reduced incubation time.
Notably, Ali Erturk’s lab of the LMU Munich has developed a 3D imaging technology to generate very high-resolution views of intact rodent organs and bodies (Cai et al. Nature Neuroscience 22, 317–327, 2018). The technology is called vDISCO and uses ChromoTek’s Nano-Boosters.

 

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Secondary Antibody Staining

For indirect labeling methods (see above), secondary antibodies are needed for detection of the primary, target-specific antibody. Secondary antibodies bind to conserved parts of the primary antibody, either in the Fc or in the Fab region. For multiplexing experiments with different primary antibodies, the secondary antibodies need to be species specific or even specific for antibody subtypes of the same species. Our secondary Nanobodies for the detection of rabbit IgG and various murine IgG subtypes are labelled with Alexa Fluor® 488, Alexa Fluor 568 and Alexa Fluor 647 dyes. They can be applied in one-step staining protocols, i.e. the simultaneous incubation of primary antibody and secondary Nanobody. Due to their small size of just 15 kDa they enable higher resolution imaging with less background. These secondary Nanobodies are called Nano-Secondaries.

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Looking for references? Please check our literature database.

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