BioSite Cationic Colloidal Gold

Technical Information

Cationic Colloidal Gold allows highly sensitive and discrete microscopical studies of anionic sites in and on cells and tissues. A simple one step incubation of sections with the gold conjugate reveals subcellular sites having net negative charge. The charge distribution may be examined with clear definition at a range of magnifications in the electron microscope by employing cationic gold of different sizes. For light microscopy a single size is employed.

Most cells of eukaryotic origin have a net negative surface charge from anionic plasma membrane components. This charge distribution is thought to be important in the movement of various macromolecules across cell walls. The role of the surface charge in cellular behavior through interaction with neighboring cells or with the intracellular matrix can now be studied microscopically with high definition by a simple one step procedure. A further great advantage of cationic colloidal gold is the possibility of performing labelling studies under various physiological conditions of pH and ionic strength.

Conjugates

The colloidal gold is made cationic by careful conjugation to poly-L-lysine, a highly positively charged amino acid chain. The conjugate is supplied in a Tris buffer containing 1% BSA and 20% glycerol at pH7. The method of manufacture allows the conjugate to be stored frozen or by refrigeration with good long term stability. Cationic Colloidal Gold is available for electron microscopical studies in ultrathin sections and for light microscopical studies in wax and resin sections. As with detection of antigens by immunolabelling, the preparation of the specimen may determine the final results. Best results for EM studies so far have been achieved with acrylic resins such as LR White.

Procedure

The conjugate is best applied in one step directly to the section according to the normal procedures for other gold conjugates. Dilution of the conjugate is normally between 1/10 and 1/100 in a buffer such as Tris or PBS adjusted to the appropriate pH. The pH will determine the degree of labelling of anionic sites. Very low pH (eg pH2-3) is likely to produce the most specific labelling with the lowest background but this must be determined experimentally. The sections should be wetted with plain buffer before applying the conjugate (as a droplet) in order to avoid non specific charge attraction. After incubating with the conjugate (1/2 hour - 2 hours or overnight) the sections are simply washed in buffer solution and then deionized water. They may then be counterstained with uranyl acetate and lead citrate and examined in the microscope. Tests should be performed to determine the effect of counterstaining on the avidity of the label since uranyl acetate is a very acid solution.

Typical Results

According to the incubating conditions (see below) sections incubated in Cationic Colloidal Gold will exhibit varying degrees of labelling in anionic sites throughout the tissue. Typically there will be intense labelling at pH7 within the heterochromatin of nuclei, along plasma membranes and within stronal components such as collagen. High labelling intensity should be expected along components of the basal lamina within epithelial cell strutures. Specific labelling for cell surface anionic sites may be determined, however, by careful adjustment of pH value during incubation, lower pH values allowing greater specificity for these sites. Non specific labelling may also be reduced by prior incubations with blocking agents such as bovine serum albumin (see below).

Controls

In order to determine specific labelling of anionic sites distinct from general background (see below) control sections should be first incubated with a solution of 1mg/ml of unlabelled poly-L-lysine in the same buffer for 1 hour before incubation with the conjugate. Controls should be incubated under similar conditions and at the same pH as the experimental sections.

A second valuable negative control is to use bovine serum albumin gold conjugate at the same dilution and pH in place of the cationic colloidal gold conjugate.

Trouble Shooting

The application of cationic colloidal gold is still in its infancy. Some suggestions are given below, however, for obtaining specific labelling at greatest intensity on thin sections. As experience increases by users of our conjugates we will issue further technical supporting information with these products.

a) No label

1. pH of solution
   The degree of labelling may be increased or decreased according to the pH of the buffer used to dilute the conjugate. Users should start with a pH of 7.0 (as supplied) and experiment either side of this value. Lower pH will reduce the total label but give greater specificity.
2. Insufficient incubation time
   Best results for most specific and most intense labelling have been found at high dilution (eg 1/100) with overnight incubations in a moist chamber (eg petri dish). Alternatively the conjugate may be used for a shorter time (eg 1/2 hour) at greater concentration, (eg 1/10 dilution).
3. Choice of embedding medium
   As with Immunolabelling, the hydrophilic resins such as LR White or Lowicryl have proved to be most successful in anionic studies of tissue at the EM level. Epoxy resins give lower labelling efficiency and greater chance of non specific charge attraction to the surface.
   For LM studies the same resins may be employed at greater thicknesses mounted on glass slides and silver enhanced. Alternatively, dewaxed sections provide good results for LM studies, in the latter case the conjugate may be diluted even further for greater specificity.
4. Choice of diluent buffer
   Buffers to dilute the conjugate may be phosphate buffered saline or Tris, as described in the instruction booklet supplied. Those buffers may be supplemented with 1% DCA to reduce non specific background.

b) High non specific labelling

1. Wrong pH
   The pH should be reduced until the background falls to very low levels but the specific labelling remains high. A series of simultaneous incubations at different pH values will indicate the optimal value.
2. Residual aldehyde groups
   Incubate for 30 min. in 0.1M ammonium chloride to block aldehyde groups remaining from the fixative.
3. Osmium tetroxide
   The use of osmium tetroxide in tissue fixation will introduce heavy metals into the tissues and may produce non specific labelling. Osmium tetroxide is best avoided.
4. Copper grids
   The use of copper grids for mounting sections may cause some change in charge distribution in solution on the sections. Use nickel or gold grids.
5. Hydrophobic charge distribution
   Non specific charge locations on the section will attract cationic colloidal particles. These may be blocked by prior incubation with 1% bovine serum albumin for 30 min. The sections should be wetted with buffer before applying the conjugate.

References:

Skutelsky E and Roth J (1986) "Cationic colloidal gold - a new probe for the detection of anionic cell surface sites by electron microscopy" Journal of Histochemistry and Cytochemistry, 34, 5, 693-696.

Vorbrodt A (1987) "Demonstration of anionic sites on the luminal and abluminal fronts of endothelial cells with poly-L-lysine gold complex" Journal of Histochemistry and Cytochemistry, 35, 11, 1261-1266.