Biology Reference
In-Depth Information
per between the closed forceps prongs but often they are no
longer suited for picking up EM grids.
12. Copper grids may form precipitates when in contact with
buffers.
13. Several negative controls are needed in order to confi rm the
specifi city of staining, especially when working with polyclonal
primary antibodies. But also monoclonal and secondary anti-
bodies may bind nonspecifi cally to the sample. The most
important negative controls are as follows (Fig. 2b ): (a) In case
of polyclonal antibody, replace primary antibody with pre-
immune serum collected from the animal before immuniza-
tion ( see Subheading 1.3 ). If pre-immune serum is not
available, use at least nonimmune (normal) serum of the same
species but this is considered not to be an adequate substitute.
(b) Replace the primary antibody with a control antibody from
the same species raised against an antigen known to be absent
in the tissue. (c) If available, use mutants lacking the antigen
or, when the distribution of transgenic proteins is studied, use
non-transformed cells or plants as controls. (d) Absorb the
primary antibody with its antigen by addition of antigen which
should abolish binding. (e) Use blocking buffer instead of pri-
mary antibody to get information about the background sig-
nal caused by unspecifi c binding of the secondary antibody.
14. Dot blots can be used to prove the presence of the antigen in
cell extracts and to detect inhibitory effects of fi xatives (e.g.,
glutaraldehyde or OsO 4 ) on antibody binding. Western blots
are required to show that the antibody recognizes a (single)
protein with the appropriate mass. It is also a good idea to fi rst
test a new antibody for immunofl uorescence where more anti-
gens are available for binding. When immunofl uorescence fails,
labeling of EM sections is unlikely to be successful. It must also
be kept in mind that antibodies suited for Western blots are not
necessarily suited for immunostaining. In Western blots anti-
bodies recognize the denatured antigen whereas in samples for
immunolabeling antigens are supposed to be in a more natural
conformation. But it is also possible that the antigen has become
inaccessible, destroyed, or extracted during preparation.
15. A small droplet of water between Parafi lm and Petri dish helps
to fi x the sheet if required.
16. Droplets placed on the intersections of a mesh-like pattern
scratched into the Parafi lm with the tip of blunt tweezers or
needles are more likely to remain in place. But be careful not
to perforate the Parafi lm!
17. Do not immerse grids into droplet. If that happens acciden-
tally, either thoroughly wash the grid in distilled water and
proceed again or continue by immersing the grid with section
side facing upwards into the droplets until the end of the
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