Immunogold Labeling of Resin-Embedded Electron Microscopical Sections - Plant Cell Morphogenesis: Methods and Protocols - page 186

procedure. If sections are collected on uncoated nickel or gold
grids, both surfaces can be stained by fully submerging grids in
reagents. Such grids can also be used for double labeling when
each side is exposed to different primary and secondary anti-
bodies (but not immersed!).
18. For prolonged incubation place covered Petri dish in a wet
chamber. Vibration of the samples during incubation helps to
shorten incubation time and may also reduce nonspecifi c
background labeling [ 1 ]. The dish of shakers may become hot
after prolonged use. A thin sheet of polystyrene beneath the
Petri dish or the moist chamber prevents heating of samples.
19. All washing steps given in this protocol are minimums! In case
of high background, staining or dirt on sections increase
number of washing steps and incubation times.
20. For easier detection at low magnifi cation, the size of the gold
particles can be increased with silver enhancement kits avail-
able from different companies.
21. This step is only required for sections with very low contrast and
when using conventional EMs without energy fi ltering [ 22 ].
Too much contrast makes recognition of small gold particles
diffi cult. Thoroughly wash grids with water after staining.

Acknowledgement

This work was supported by the Austrian Science Fund (project
no. P 22957-B20 to IF).

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