Biomedical Engineering Reference
In-Depth Information
Table 1
Summary of quantum dot-labeling protocol for neurons and glia
Preprocessing and fi xing
Remove media from wells by gently aspirating
Wash cells with warmed PBS
Fix cells with 4% paraformaldehyde (Electron Microscopy Sciences, catalog #157 15-S) in
PBS for 10 min at room temperature
Wash cells 3× with PBS
Permeabilize cells with 0.2% Triton X-100 (Fisher Scientifi c, catalog #BP151-100) in PBS
for 5 min
Wash cells 3× for 5 min with PBS
Incubate with 10% horse serum in PBS for 30 min at room temperature
Rinse with PBS
Apply Streptavidin/Biotin Blocking Kit (Vector Labs, catalog #SP-2002)
Primary incubation
Rinse with PBS
Add biotinylated molecule of interest (e.g., antibodies; use ProtOn Biotin Labeling Kit or
similar for biotinylation; Vector Labs, catalog #PLK-1202)
Incubate for 2 h at room temperature (biotinylated secondary antibody for 1 h - alternative
three-step-labeling protocol)
Remove antibodies by gentle aspiration and rinse 3× with PBS
Quantum dot incubation
Add streptavidin-conjugated quantum dots (we used Quantum Dot Corporation's 605-nm
quantum dots here, catalog #1010-1) in 10% horse serum
Incubate for 1 h at room temperature
Rinse 3× with PBS
Mount with 90% glycerol (Sigma, catalog #G-6279) in PBS
Reproduced from Pathak et al. (
2006
)
neural cells incorrectly due to nonspecifi c putative electrostatic interactions. We
observed this when conjugating antibodies directly to quantum dots, which
resulted in unconjugated quantum dots nonspecifi cally staining the nucleus of
Muller cells (see Fig.
2i
). Nonspecifi c binding was also observed when using
other published protocols for nonneural cells (Wu et al.
2003
) . Blocking condi-
tions also need to be carefully optimized since most standard blocking approaches
did not work satisfactorily in our hands, including 1-5% bovine serum albumin,
10% horse serum, and 10% fetal bovine serum among others, which resulted in a
high level of nonspecifi c quantum dot binding to the cells (data not shown).
Another advantage to labeling with quantum dots is that each individually visual-
ized dot in a fl uorescence micrograph represents one to three individual quantum
dots based on our own calculations and those of others (Chan and Nie
1998
) . This
means that qualitative and potentially quantitative information can be measured
for individual binding events between quantum dot-conjugated molecules and
their cellular molecular targets, a direct result of the underlying physics (Michalet
et al.
2005
; West and Halas
2003
) that cannot be done with standard ICC (see
Dahan et al.
2003
, for an example).
