SPI-Mark™ Cationic Colloidal Gold Conjugates
Procedure
Procedure
The gold conjugate is best applied in one step directly to the section (LM
or EM) according to normal procedures for other gold conjugates. Dilution of the
conjugate is normally between 1/10 and 1/100. This may be in water or a
buffer such as PBS or TBS as appropriate. The pH will determine the
degree of labeling of anionic sites but too high an ionic salt concentration
in the buffer may also affect the charge on the specimen Tests should be
performed comparing dilutions in water and various strengths of buffer at
the appropriate pH. Very low pH (e.g. pH1-3) is likely to produce the most
specific labeling with the lowest background but this must be determined
experimentally.
After incubating with the conjugate (15min - 2 hours or overnight) the
sections are simply washed in deionised water. For LM sections silver
enhancing is used according to the protocol provided with the
SPI-Mark Silver Enhancement Kit.
Typical enhancing times are 5-10 minutes for 5nm and 5-20 minutes for
1nm conjugates. The sections may then be counterstained with hematoxylin
and eosin as normal. For EM sections the gold signal may be examined after
counterstaining with uranyl acetate and lead citrate. Tests should be performed to determine
the effect of counterstaining on the avidity of the label since uranyl
acetate is a very acid solution. If desired the gold particles may be
enlarged by simple enhancing in the SPI-Mark Silver Enhancement Kit
for a few minutes.
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