SPI-Mark™ Colloidal Gold Reagents
Specific Immunolabeling Protocols
Double labeling of EM Sections
(I) labeling both sides of the section
In this method the first antigen is labelled on the upper side and the second
antigen on the lower side of the section. Use uncoated grids to mount
sections. Perform all incubations by floating sections on droplets to avoid
contaminating the upper side.
(A) Follow steps 5.1 (a - e) above using the first primary antibody.
(B) Optionally coat the reacted side of the section with carbon evaporation.
(C) Turn the section over and repeat steps in 5.1 (a - e) using the second
primary antibody but also using a different gold particle size for the second
gold labelled antibody. Typically this means using 5nm for the first side and
15nm for the second side.
Because two different sides of the section are incubated independently the
two primary antibodies may be raised in the same animal species.
Optional silver enhancement:
(D) If required either side of the section may be silver enhanced. Place the grid
on drop of freshly mixed
silver enhancing solution for a period
of 5-10 minutes. Determine the time experimentally
Alternatively, the same particle size may be used on both sides of the section
and one side enhanced with silver to produce a different final size for double
labeling.
(E) Wash thoroughly in distilled water to stop the reaction.
(F) Counterstain if required with
uranyl acetate and
lead citrate
as normal. Wash and examine in the electron microscope.
(II) labeling the same side of the section
In this method both antigens are labelled on the same side of the section using
different particle sizes. The tissue section may be mounted on a Formvar
coated grid for 1 incubations.
(A) Follow steps 5.1 (a - e) above using the first primary antibody (e.g. from mouse).
(B) Repeat steps 5.1 (a - e) above but using a second primary antibody from
different species (e.g. from rabbit) with the appropriate gold labelled antibody
(e.g. Goat anti-rabbit).
Optional silver enhancement:
(C) The section may be silver enhanced after step (a) above in order to grow
particles to any size required before performing the second incubation
procedure. Follow steps 5.1 (f - g). Do not expose the section to the electron
beam before the second immunolabeling procedure since this will denature all
proteins.
Counterstain:
(D) Counterstain if required with uranyl acetate and lead citrate as normal.
Frozen sections may be stained with osmium tetroxide vapour. Wash and
examine in electron microscope.
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Thursday May 17, 2012
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