UC San Diego
School of Medicine
Cellular & Molecular Medicine 
Electron Microscopy Facility
 
 

This method is used for subcellular (co-) localization of antigens in cells and tissues at the EM level.



Instead of using resin-embedded samples, we prefer to use cryosections for IF or EM immunolabeling experiment. Although they are more difficult to section at a consistent thickness, the detection of antigens in thawed cryosections is better than using resins. Any success using an antibody on semithin and/or ultrathin cryosections will depend on the sample fixation and the antibodies used. Labeling generally works better if the antigen in question is reasonably abundant. 



Immuno-Labeling of Ultra-thin Cryosections:


Immuno electron microscopy on ultrathin cryosections is usually much more labor intensive than morphology (plastic-embedded) samples, and it is extremely difficult to predict or guarantee good results. The morphological detail is mostly lost, and the overall appearance is very different from plastic sections. Nevertheless, when good results are obtained, an immuno-EM image can be a powerful localization tool. Using secondary reagents conjugated to gold particles of different sizes, two different antigens can be (co-) localized within the same sample sections.



Antibodies to be used for semi-thin IF and/or immuno-EM:


Depending on the fixation / accessibility of the antigen and the affinity & purity of the primary antibody, the detection limit / sensitivity may be extremely low. When no clearly specific label is found it does not mean that no antigen is present. Conversely, when there is some label, there probably is a reasonable amount of antigen molecules present.


In general, a crude serum fraction of a particular antibody will not be the best form to use. Purified forms typically produce cleaner results with less non-specific background on cellular structures or cytoplasm. In most cases, good to better to best results may be obtained using caprylic acid, protein A or affinity purification respectively. Purified antibodies with a concentration of 1 to 5 mg/mL will be optimal.


Typically, if an antibody works well for western blotting at a 1/1000 dilution it may work at a 1/100 to 1/250 dilution for IF (on semithin cryosections) and at 1/10 to 1/100 for immuno-EM.


However, due to the difference in thickness of the sections (and hence the difference in the amount of antigens that are accessible) an antibody that works well for IF use on semithin cryosections may not work at all at the immuno-EM level.


GFP & Other Tagged Proteins:


We have successfully used antibodies to some very common molecular epitope tags, such as Flag, GFP, HA, myc, and V5 in immuno-EM. Usually there is none, or very low non-specific cross reactivity of these antibodies with “natural” components of the sample. When a construct is used, the transfected cells usually overexpress the (tagged) protein, and this also makes detection by immuno-gold technique more favorable, and more likely to be successful. Even overexpressing a non-tagged construct can dramatically improve the chances of detection, in comparison to trying to label the endogenous form.


Gold Particles:


Colloidal gold particles are available in different sizes, and the gold can to be coupled to a biologically relevant molecule.


The detection reagents can either be a colloidal gold-conjugated Ig fraction (of a primary or secondary antibody) or a gold-conjugated molecule that binds to specific targets. For instance, gold-conjugated protein-A will bind with high affinity to rabbit-IgG's. 


Double labeling experiments using differently sized gold particles can be done either by using two different species of primary antibodies in combination with their respective secondary gold-conjugated antibodies, or by using protein-A gold conjugates of different sizes, in which case it is possible to use two primary antibodies of the same species (rabbit). A fixation step destroying the protein-A binding epitope and/or "(gold-) free protein-A" can be used to prevent cross detection of the different primary IgG’s.

Immuno-EM; Gold Labeling of Ultra-thin Cryosections