SPI-Mark™ Cationic Colloidal Gold Conjugates
Trouble shooting
The following suggestions are made to counter some more frequent observations
in the use of cationic colloidal gold.
No label
pH of conjugate
The degree of labeling may be increased or decreased according to the
pH of the buffer used to dilute gold conjugate. Users should start with
pH2 for greatest specificity and experiment either side of this.
High salt content
The strength of the buffer used to dilute the gold conjugate may affect the
labeling. High salt content effectively compress the charge at the ionic
sites and on the gold particles, making charge attraction less efficient
Dilution of the gold conjugate in water at the correct pH may indicate this
problem. The diluent should contain 1% BSA
to reduce non specific background.
Insufficient incubation time
Best results for most intense labeling have been found at high dilutions
(e.g. 1/100) with overnight incubations in a moist chamber such as a petri
dish. Alternatively the conjugate may be used for a shorter time (e.g. 15
minutes) at a greater concentration (e.g. 1/10). Higher background
labeling may be caused by high concentrations.
Choice of embedding medium
As with immunolabeling, the hydrophobic resins such as
LR White and
Unicryl have
proved most successful in anionic studies of tissue at the
EM level. Epoxy resins, in
part because they require complete dehydrations, give lower labeling efficiency and greater chance
of non specific charge attraction to the surface. For LM studies the same
resins may be employed at greater thickness mounted on glass slides.
Alternatively cryo sections or paraffin wax sections may be used.
High Background
Wrong pH
The pH should be reduced until the background falls to very low levels
but the specific labeling remains high. A series of simultaneous
incubations at different pH values will indicate the optimal value.
Residual aldehyde groups
Incubate with 0.1M ammonium chloride for 30 min to block aldehyde
groups remaining from the fixative. In addition incubate with 1% BSA
before the application of cationic gold conjugate and include 1% BSA in
the incubating diluent.
Osmium tetroxide
The use of osmium tetroxide in tissue fixation will introduce heavy metal
into the tissue and may produce non specific labeling.
Osmium tetroxide is best avoided.
Copper Grids
The use of copper grids for mounting EM sections may cause some
change in charge distribution on the sections. Furthermore, for low pH protocols,
the copper grid will actually start to dissolve. Nickel or gold grids would
be a much better choice.
Hydrophobic charge distribution
Non specific charge locations on the section will attract cationic gold
particles. These may be blocked by the inclusion of 0.1%
Tween® 20 in the
incubation solution.
Section mounting (LM).BR>
Glass surfaces may attract cationic gold. Blocking with 1%
BSA and the
inclusion of 1% BSA in the incubating buffer will reduce this source of
background.
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