Quantum Dots
Quantum Dot Protocol
To create a quantum dot that will tag AMPA receptors choose a quantum dot conjugated to an IgG antibody that also has a fragment antigen-binding region (aka F(ab’)2 or Fab fragment, the part of an antibody that binds antigens). This entire construct, QD-IgG-Fab, is your secondary antibody construct and the IgG and Fab are usually from different host animals. This is then incubated with primary antibody fragment (Fab) that is specific to the protein of interest, in this case AMPA receptors. 
An example experimental protocol might look like this: A total of 1 μL of 655-nm Quantum dots (Qdots) conjugated to goat (Fab‘)2 anti-mouse IgG (Invitrogen) were incubated with 1 μL Fab anti-GluA2 in 7 μL PBS for 20 min at room temperature. Nonspecific binding was blocked by adding 1 μL of 10% casein stock solution for 15 min (Vector Laboratories), and this solution was kept at 4 °C throughout the experiment. Neurons were incubated for 10 min at 37 °C in 1 mL culture medium containing 1 μL of the anti-GluA2-coated Qdot solution, then rinsed and mounted in an aluminum chamber containing Tyrode solution (30 mM glucose, 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25 mM Hepes) on a Nikon microscope (NIKON Eclipse TE2000-U) thermostated to 37 °C using an air blower (World Precision Instruments) and an objective heater (Bioptechs). Single Qdots were detected through a100 × 1.4 N.A. oil immersion objective, using a 100-W mercury lamp and appropriate excitation/emission filters (Chroma). Sequences of 50 s, corresponding to stacks of 1,000 images with an integration time of 50 ms, were acquired using a CCD camera (Quantem; Roper Scientific). For each coverslip, Qdots were followed on randomly selected dendritic regions (size 20 × 20 μ m) for up to 20 min. Two coverslips of each condition were processed for a total of 3 – 4 neuronal cultures. We checked for specificity of Qdot binding by preparing primary hippocampal cultures from GluA2 KO mice dissected at p0. Anti – GluA2- conjugated Qdots bound minimally to cultures from GluA2 KO compared with those prepared from wild-type littermates.