October QDot Update

October 5, 2020

by Thomas Porter

After a long optimization process, the conjugation of DNA to primary antibodies seems to be working but the labeling process is not. The conjugated antibodies were used to label actin in fixed cells. While the positive control of adding fluorescent secondary antibodies (shown in figure 1) worked well, the DNA cages were not able to be seen.

Figure 1. Positive control staining for actin in fixed cells.

It was determined that the micelles that contain the dye at the heart of the cages were not bright enough. We are still attempting to fix this problem since currently the micelles are crashing out of solution faster than normal. We believe that either the polymer or the dye needs to be replaced.

We are also working on our next steps of labeling tissue using the DNA cages. We started with a solution of DNA cages with indigo dye encapsulated in the micelles. These give a blue color that should constrast against the tissue slices. Initially, we simply added the solution of cages to the tissue slices in hopes that they would stick. Figure 2 shows the results of this study so far. The cages do appear to be staining to some degree, but we really want a more concentrated dye either by increasing the loading in the micelles or concentrating the cage solution even more.

Figure 2. Tissue slices stained with indigo DNA cages. The different panels are different dye loadings of the micelles before caging. A) 0.5 mg/ml indigo B) 1 mg/ml indigo C) 2 mg/ml indigo

Our next steps are:

1. Continue working on the encapsulation of the fluorescent dye in order to move forward with fixed cell labeling

2. Increase the dye loading for the indigo particles

3. Try different colored dyes for the non-specific tissue labeling

-Elizabeth