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Antibody detection
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Individual antibodies
With commercially available, fluorescent-labeled antibodies, the manufacturer's instructions regarding antibody concentration for in situ procedures should be followed.As a general guidline, a 1:100 dilution in 4xSSC of a 1 mg/ml IgG stock solution should work well, after 5-10 minutes incubation time at 37 C. For each slide, 1 µl antibody stock is mixed with 100 µl 4xSSC right before use.
Every time a new antibody is purchased, it should be tested at different dilutions, to find the optimal range for that antibody. Some antibodies perform well even at a 1:500 dilution whereas others do not. Many commercially available antibodies are sold in 0.5-2 ml volumes as stock solutions, usually at 0.5-1 mg/ml, which should be sufficient to detect thousands of slides. It is much more economical to buy concentrated stocks of antibodies than antibody solutions already diluted for direct use.
In order to stabilize the antibody soution for long-term storage, one can add BSA to the antibody (final concentration about 10 mg/ml) or keep the antibody just like an enzyme, in 50% glycerol. Stock solutions should always be stored at -20 C.
Antibody combinations
Antibody combinatorial schemes allow, for example, triple color detection and signal amplification for two or more probes labeled with different haptene-dUTPs. Fluorophores are chosen so as to match the filters with which the fluorescence microscope is equipped. When detecting the haptenes, one can chose only one fluor-labeled antibody for each probe (#2 in figure below) or one can use primary, secondary and even tertiary antibodies to amplfify the signals (#1 in figure below). When combining antibodies in multicolor detection schemes (such as the colored figure below, identical with Table 7), it is important to pay special attention to potential, unwanted antibody-antibody interactions or cross-reactions. It is, also useful to purchase antibodies previously affinity-purified for interactions with other animal species used. To illustrate the importance of this concept, the detection scheme in the figure below uses antibodies from different animals to achieve simultaneous triple color staining and signal amplification of three FISH probes, each labeled with a different haptene (BIO, DIG and DNP). Because of the various antibody sources, the detection protocol was divided into five different steps. Otherwise, there would have been unwanted cross-reactions. For example, if mouse antiavidin-BIO not goat were used at step II, it would have been recognized by the sheep antiM-Cy3 when mixed in the same vial. Similarly, goat antiavidin-BIO could not have been used at step IV, as the donkey antiG-DTAF from step V would have recognized it, just as well as the goat antiR-FITC.
Signal amplification
Antibody combinations allow signal amplification in FISH. Figure below shows that digoxigenin-labeled DNA can be detected with a varitety of labeled antibodies (using different fluors) and that the signal can be further amplified using secondary and tertiary antibodies. The colors depict the approximat color of the respective fluorophore.
Research or commercial DNA probes sometimes provide only a weak FISH signal. When labeled with haptene-dUTP (BIO, DIG, DNP), the fluorescent signal is usually provided by a fluor-labeled antibody (primary antibody) against the haptene. To amplify the signal, another fluorescent-labeled antibody (secondary antibody) against the primary antibody is used.
When a DNA probe is labeled by a fluor-dUTP, signals are usually weaker than the signal yielded by the same probe if it were detected with an antibody. The reason is that an antibody molecule carries 3-4 fluors attached to it. The weak signal of a fluorescent-labeled probe can sometimes be amplified using antibodies raised against that fluor molecule. Antibodies against fluorescein, rhodamine, Texas Red, Cascade Blue and other fluors are available commercially. These primary antibodies will bind to the respective fluor, and a secondary antibody labeled with the same fluor, will be used to bind to the primary antibody and amplify the signal. This is illustrated in Fig. 9j and 9k, where a FITC (green) fluorescent signal was amplified using a rabbit anti FITC IgG, followed by a FITC-labeled goat anti-rabbit IgG.
The same fluor does not necessarily have to be used. For example, a rhodamine signal can be amplified by using a primary anti-rhodamine antibody followed by a secondary antibody labeled with Cy3 (rhodamine and Cy3 have similar emission spectra) or any other dyes.
Other signal enhancement procedures: the tyramide detection system (TSA)
A sometimes useful procedure for enhancing FISH signals is the Tyramide Signal Amplification (TSA), provided by NEN Life Sciences. In this procedure, after usual hybridization, an antibody conjugated to horseradish peroxidase (HRP) is bound to the labeled DNA probe. HRP catalyzes the deposition of biotin- or fluor-labeled tyramide on the slide. This precipitation step allows many molecules of labeled tyramide to precipitate on the slide where the HRP is located, and thus provides a strong FISH signal. The precipitation, however, makes the signal