Biology Reference
In-Depth Information
studies is relatively new, a significant number of primary and secondary
conjugates of quantum dots with varying sizes and emission wavelengths are
commercially available. Furthermore, quantum dots with reactive linkers are
also available for producing direct conjugates of interest, making the dots
a convenient, adaptable, and flexible choice as probes for molecules and
molecular interactions. As above, the protocol described applies to bacteria and
cells adsorbed to a substrate such as silicon chips or Thermanox® cover slips.
If used with pellets or suspended material, the methods can be modified accord-
ingly. For some applications, it may be advantageous to pre-fix the samples
to prevent progression of biological processes during labeling. When labeling
live material, active processes such as internalization, intracellular trafficking,
and cell division can be controlled as needed with temperature or suitable
chemicals such as azide or other metabolic inhibitors. Using quantum dots,
common steps such as titrating antibodies, conjugates, and blocking reagents to
optimize labeling and detection of targets by fluorescence microscopy generally
provide optimal electron imaging as well. Thus, fluorescence microscopy is
useful for rapidly assessing the specificity and sensitivity of the labeling exper-
iment before the samples are processed for electron imaging. However, this
is not necessarily the case when detergents or solvents are used to perme-
abilize the cells or bacteria, as cellular ultrastructure can be affected adversely
and dramatically by such agents, without noticeable detriment
in a light
microscope.
3.3.1. Pre-Fixation (Optional)
1. Wash samples twice with an appropriate physiological buffer such as PBS or
HBSS to remove residual medium components.
2. Immerse the samples into PBS containing 4% paraformaldehyde and 0.1%
glutaraldehyde. Incubate at room temperature for 15-30 min.
3. Wash sample twice for 15 min with buffer.
3.3.2. Immune Labeling
1. Block the samples by immersing in PBS containing 2% (w/v) globulin-free BSA.
Incubate at room temperature for 30 min.
2. Replace the blocking solution with primary antibody such as immune or pre-
immune mouse serum diluted to an appropriate concentration in blocking buffer.
Incubate the mixture for 30 min at room temperature or overnight at 4 °C. Deter-
mining the optimal concentration and length of incubation may require empirical
experimentation.
3. Wash the samples at least twice for 15 min in blocking buffer.
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