BioSite Cationic Colloidal Gold
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
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.
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.
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
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).
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.
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
- 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.
- 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).
- 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
good results for LM studies, in the latter case the conjugate may be
diluted even further for greater specificity.
- 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
- Wrong pH
The pH should be reduced until the background falls to very low
levels but the specific labelling remains high. A series of
at different pH values will indicate the optimal value.
- Residual aldehyde groups
Incubate for 30 min. in 0.1M ammonium chloride to block aldehyde
groups remaining from the fixative.
- 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.
- 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.
- 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.
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.