The principle of the immunolocalization technique in plants basically involves fixation and permeabilization of cells, the use of monoclonal or polyclonal antibodies attached to a signaling molecule, usually a fluorochrome and detection of the target molecule by using an epifluorescence microscope.

1. Immunolocalization

2. Definition
Immunlocalization: Technique using specific antibodies to localize
macromolecules (proteins, polysaccharides) within biological material
(subcellular fractions, cells, tissues, biofilms).

3. Introduction
I. based on the detection of specific targets (“antigens”; mostly
proteins or polysaccharides) in biological samples (cells, tissue,
biofilms) by antibodies as probes.
II. Regularly, antibodies of IgG subtype and IgM and IgA subtypes are
used.
III. The antibodies may be monoclonal or polyclonal.
IV. Specific non-covalent binding is brought about by the hypervariable
region of the antibody (paratope), exactly fitting to an epitope.
V. The hypervariable regions are the tip parts of the Y-shaped arm of
the antibody

4. • All other parts, including the Fc-part, are structurally identical within
one antibody subclass. Localization of the antigen is only possible
when the specific (or primary) antibody (“probe”) binds to a marker
• A variety of antibody-marker systems are available for different
applications The marker may be coupled by different methods to the
probe

5. The marker may be coupled by different
methods to the probe!
• By direct coupling of the marker to the probe prior to use in a localization
experiment. The detection procedure requires just one binding step. The method
is also termed direct immunolocalization.
• By binding of a secondary probe (mostly a secondary antibody) to the Fc-part of a
primary antibody after this primary antibody is already bound to the specific target
(two-step procedure). The secondary probe is coupled to the marker. The method
is also termed indirect immunolocalization.
• By binding of tertiary probes to the secondary probe(for signal amplification).
Tertiary probes are responsible for binding or deposition of the respective marker

6. Immunolocalization in light and electron
microscopy
• For immunolocalization in (fluorescence) light microscopy, fluorescent dyes are used as markers
(immunofluorescence microscopy).
• The marker consists of the dye covalently coupled to a protein (e.g., the secondary antibody) that
binds to the primary marker, the antigen-specific antibody.
• It is also possible to detect more than one target in a sample by the use of marker-probe systems
with specificities for different targets coupled to fluorescence markers with discernable
absorption–emission spectra.
• Access to the antigen in tissues or embedded samples can be achieved by (cryo-) sectioning or by
permeabilization.
• For (nonfluorescent) bright field microscopy, marker systems that catalyze enzymatic reactions are
used, leading to deposition of dark-stained precipitates at the antigen binding sites.

7. Immunolocalization in light and electron
microscopy
• For electron microscopy, probes are coupled to colloidal gold as an electron dense
marker.
• As stated for light microscopy, the marker can be directly coupled to the primary
antibody, but regularly, a secondary system is combined with a primary antibody.
• Also, tertiary systems for signal amplifications are used. In equivalence to multiple
detections of targets in light microscopy, it is possible to detect more than one
target in a sample by use of marker-probe systems with discernable particle sizes
of gold colloids.
• Particle sizes between 5 and 25 nm are used for experiments at the cellular level.
Small gold particles may also be enlarged with silver or gold salt solutions for
easier localization. For epitope mapping in macromolecules, 1-nm markers
(“nanogold”) are available

8. Immunolocalization in light and electron
microscopy
• Several modifications of the original immunogold marker technique can increase
the resolution and sensitivity of the method.
• Dodecaborane clusters may be coupled to antibodies or Fab-fragments via
polylysine dendrimers.
• The boronated antibodies have a lower detection limit in immunolocalization
experiments than conventional gold conjugates.
• Detection of boron is performed by elementspecific imaging in energy-filtering
transmission electronmicroscopes

9. Immunolocalization in light and electron
microscopy
• Techniques like catalyzed reporter deposition (“CARD”) use tertiary markers. CARD
is a technique for amplification of a signal, originally designed for Western blotting,
but modified for light and electron microscopy.
• In the first step, a specific primary antibody is coupled to the target. Then, in a
second step, a secondary, biotin-tagged antibody is coupled to the primary
antibody.
• A streptavidin–peroxidase conjugate is then bound to the biotin tag. Peroxidase
catalyzes the deposition of tyramides indirectly.
• The tyramide itself is bound to a detectable marker (e.g., again biotin), and may be
localized by the use of streptavidin-coupled colloidal gold for detection in the
electron microscope
• For light microscopy, fluorescent dye-coupled tyramides are used.