SPI-Mark™ Colloidal Gold Reagents
Specific Immunolabeling Protocols
Pre-embedding labeling of Cell Surface Antigens for EM
(A) Isolate cells from tissue with trypsinisation or separate blood cells by
gradient centrifugation. Spin to a soft pellet and re-suspend in subsequent
incubation solutions.
(B) Prefix cells in
0.1% glutaraldehyde or
2-4% paraformaldehyde
in depending on antigen sensitivity.
(C) Wash 3 times in PBS containing 50mM glycine to quench aldehydes.
(D) Block cells for 30 min in PBS + 1% BSA+ 10% normal serum (second antibody species).
(E) Wash twice with PBS + 1% BSA.
(F) Incubate with primary antibody diluted in
Buffer A
for 1 hour (typically 1/100 for a monoclonal)
(G) Wash twice in Buffer A.
(H) Incubate in second gold labelled antibody diluted in Buffer A for 1 hour (typically 1/10 - 1/100).
(I) Wash twice in PBS
(J) Fix in 1% glutaraldehyde in PBS for 15 min.
(K) Wash twice in distilled water.
Optional silver enhancement:
(L) Incubate in fresh
silver enhancing solution
for 5-10 min. This is determined experimentally.
(M) Wash thoroughly in distilled water to stop the enhancement and to remove
silver ions from the cells.
(N) Spin to a firm pellet, continue through to dehydration and embedding for
EM or LM investigations. Counterstain as normal.
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Thursday May 17, 2012
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