Biology Reference
In-Depth Information
8. Scissors.
9. Grid mat or grid holder pad.
10. Ultrathin sections collected on nickel or gold grids (
see
Note 12
).
3
Methods
Work in a dust-free clean environment and avoid contamination of
solutions, sections, and material. Wash forceps in distilled water
between each step. Cover Petri dish between handling of grids in
order to reduce evaporation. Never let grids completely dry out
before the end of the incubation procedure! It is advisable to run
the necessary negative controls concurrently in the same or another
Petri dish (
see
Note 13
). Before immunolabeling a number of posi-
tive controls should be made (
see
Note 14
).
1. Place a sheet of Parafi lm on the bottom of a Petri dish (
see
Note 15
; Fig.
2a
). Be careful not to touch the surface of the
Parafi lm with ungloved hands.
a
c
d
b
BB
pAB
BB BB
BB
BB sAB BB BB
BB
Ad Ad
BB
PIS
BB BB
BB BB
sAB BB BB
BB
Ad Ad
BB
BB
BB BB
BB
BB sAB BB BB
BB
Ad Ad
Fig. 2
Immunogold labeling of EM sections. (
a
) Petri dish with Parafi lm onto which a mesh-like pattern was
engraved using a blunt needle. (
b
) Series of incubation steps for immunogold labeling and two important nega-
tive controls (
see
Note 13
). The sections on the grid in the upper row (now in banding buffer BB) will be incu-
bated in primary antibody (pAB). The sections on the grid in the middle row will be incubated in pre-immune
serum (PIS) to check specifi city of primary, polyclonal antibody. The sections on the grid in the lower row will
be incubated in blocking buffer to check specifi c binding of the secondary antibody (sAB). All other steps are
the same. Ad = distilled water. (
c
) Removal of excess liquid by vertically touching fi lter paper with grid. (
d
) A
wedge of fi lter paper is used to remove liquid between prongs of tweezers
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