SPI-Mark™ Colloidal Gold Reagents
Specific Immunolabeling Protocols
Post Embedding labeling Tissues for EM
(A) Place the grid, section downwards, on a 50µl droplet of 10% heat inactivate
normal serum (of the gold labelled animal species, e.g. for Goat anti-rabbit:
gold use normal goat serum) in
TBS or PBS buffer
and incubate for 10 min.
(For Protein A, Protein G or Protein A/G gold conjugates omit this step).
(B) Without washing, gently blot off excess and transfer the grid to the surface
of a 50µl droplet of primary antibody diluted in buffer A (above). Incubate for 30
min to overnight.
(C) Transfer the grid to a series of 5 or more 50µl droplets of
Buffer A, leaving 5
min on each, to remove unbound antibody.
(D) Transfer the grid to two successive 50µl droplets of gold conjugate diluted
in the appropriate buffer A. The first drop will remove excess buffer and the
second drop will provide the incubation. Incubate for 1 - 4 hours.
(E) Transfer the grid to a series of 50µl droplets of distilled water (5 x 2 minutes
or more) to remove unbound gold conjugate.
Optional silver enhancement:
(F) If required place the grid on a drop of freshly mixed
silver enhancing solution
for a period of 5 - 10 minutes. Determine the time
experimentally beforehand by enhancing gold particles which have been
allowed to dry on a Formvar® coated grid.
(G) Wash thoroughly in distilled water to stop the reaction.
(H) Counterstain if required with
uranyl acetate and
lead citrate as normal.
Frozen sections may be stained with
osmium tetroxide vapour. Wash and
examine in the electron microscope.
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Thursday May 17, 2012
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