SPI-Mark™ Colloidal Gold Reagents
Trouble shooting (microscopy)
Clustering
Clustering described here should not be confused with amplification produced
with indirect labeling. In the amplification process each primary antibody may
have attached several gold labelled secondary antibodies. This will lead to
small groups of gold particles, perhaps 2-4, which surround single antigen
targets in the tissue section.
For LM Grade and BL Grade conjugates clustering is of less importance since
the particles are not seen individually in the LM sections or on blotted
membranes. Nevertheless excessive clustering during manufacture is strictly
avoided since this would otherwise lead to long term instability. Excessive
clustering may sometimes be seen on EM sections for the following reasons.
(A) Clumped primary antibody. Use fresh antisera. Microfilter if possible to
remove bacteria.
(B) Contamination of the gold conjugate. Caused by lysine or other protein
additives, by thimerosal or mercaptoethanol. Conjugate stored at extreme pH.
Conjugate overheated. Store at 4°C in separate aliquots in the original buffer
supplied until required for use. Do not freeze and thaw frequently. Do not place
pipettes into a single vial frequently.
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Thursday May 17, 2012
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