Quantum Dot Labelling

Reproducibility is key to scientific research. It is, in fact, a hallmark of the Scientific Method, controlling variables until the same result can be achieved time again. There are few things more frustrating than following a simple experimental protocol and not achieving the predicted result. This inevitably leads to wasted time, wasted reagents, and very frustrated researchers.

At least some of these frustrations could be avoided if more people made their results (both negative and positive) openly available. It is for this reason that I have decided to share a recent experience of quantum dot (Qdot) labelling on this blog.

In my current research project, I am trying to label a nuclear hypoxia inducible transcription factor (HIF-2α) using immunofluorescent methods and a Qdot labelled secondary antibody (A-10195: F(ab’)2-Goat anti-Mouse IgG (H+L) Secondary Antibody, Qdot 625 conjugate). The basic protocol can be found on the Thermo Fisher Scientific website. Since Qdot-conjugated secondary antibodies are large and therefore have difficulty in accessing the nucleus, the standard immunocytochemistry protocol was optimised by reducing fixation and increasing permeabilisation.

Despite using a suggested positive control of mouse anti-beta tubulin primary antibody to begin with and an optimal 39106-AT-QDot 625 filter set (Chroma Technology Corporation, USA) to obtain the best possible signal, there was large amounts of non-specific staining in HeLa cells.

No primary_QD1in500_red
Fixed HeLa cells labelled with the Qdot-conjugated secondary antibody only, no primary antibody

 

No primary_QD1in500_brightfield
Corresponding brightfield image of fixed HeLa cells

 

I am confident that the primary antibodies are good, because I have used them with multiple organic dyes (Alexa Fluors and cyanines) and that the Qdot-conjugated secondary antibody (<6 months old) has been stored correctly at 4°C.

 

Tubulin_1in100_Cy3_1in500
Fixed HeLa cells immunolabeled with an anti-beta tubulin primary antibody and a Cy3-conjugated secondary antibody

 

SC35_1in100_and_Cy3_1in500
Fixed HeLa cells immunolabeled with an anti-SC35 primary antibody and a Cy3-conjugated secondary antibody

After contacting Thermo Fisher Scientific Technical support with images of my Qdot labelling, who were very helpful and patient in their attempt to diagnose the problem, it was agreed that the Qdot-conjugated secondary antibody was indeed showing unusual non-specific staining. One response suggested that the cause of the non-specific binding might be due to:

“…the mounting medium that will quench the Qdot signal as Qdots are sensitive to copper and heavy metal traces often found in mounting medium”.

I therefore repeated the Qdot staining without any mounting medium, but achieved similar results: non-specific binding to HeLa cells, even with the Qdot-conjugated secondary antibody only and no primary antibody. Having repeated variations of the protocol on numerous occasions, it appears that even nuclear structures such as the splicing factor SC35 yield staining that resemble tubulin-like structures?!

SC35_1in100_QD_1in500_red
Fixed HeLa cells immunolabeled with an anti-SC35 primary antibody and a Qdot-conjugated secondary antibody
SC35_1in100_QD_1in500_brightfield
Corresponding brightfield image of fixed HeLa cells

Thermo Fisher Scientific support concluded that it must be an isolated issue and that they were prepared to replace the Qdot-conjugated secondary antibody or provide credit on my account. As I do not wish to try my luck with another Qdot-conjugated secondary antibody from the same batch, I opted for an alternative Qdot-conjugated secondary antibody, from the same company, but a different host species…fingers crossed that this will be more successful.

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