Detection

The choice of the detection scheme depends on several factors: (i) the number of haptens and fluorochromes used for probe labeling; (ii) the number of antibody layers required to obtain a sufficiently strong signal; (iii) the colour of nuclear counterstain. Some probes show high hybridization efficiency and require only one detection layer. We even have good results using directly labeled probes for small single copy sequences. The more different labels you want to detect in parallel the more difficult is the procedure to get sufficient stains. One should also plan carefully the detection scheme in advance in order to avoid reactions between antibodies used for the detection of different fluorochromes. In our lab different combinations of up to five or six different fluorochromes were successfully tested with regard to efficient signal intensities and distinct colour separation using a Leica SP2 confocal microscope. For a five colour detection scheme we got best results using Alexa488 (FITC) in parallel with Cy3 (TAMRA), Texas Red, Cy5 (Alexa633) and DAPI as DNA-counterstain. In addition Alexa514 can be implemented, however this in this combination this fluorochrome requires unmixing software for a clear delineation of all colours. In this combination we normally use direct fluorochrome labeling for TAMRA and TexasRed (and optionally FITC) while Alexa514, Cy3 and Cy5 should be detected using the respective conjugates with appropriate antibodies. To our experience in principle all commercially available fluorochrome-conjugated antibodies from the established companies work well. However, one should be aware that the quality of an antibody can vary sometimes depending on the batch provided.

Simultaneous detection of six colours was done by adding Alexa514 to the detection scheme above. Alexa514 is a very bright fluorochrome. It has a broad emission spectrum detectable in the emission spectrum of Alexa488 as well as in settings for Cy3-channel. Therefore the Alexa514 requires unmixing or substraction of the respective channels (see figure 9).

1. After hybridization, peel off rubber cement and strip off the coverslip and transfer it to 2xSSC;

2. Wash 3x5 minutes in 2xSSC at 37°C, shaking;

3. Wash stringently 3x5 minutes in 0.1xSSC at 60°C, shaking;

4. Rinse briefly in 4xSSC/0.2%Tween;

5. Block in 4xSSC/0.2%Tween + 4% BSA (bovine serum albumin) for 10-15 minutes at 37°C;

6. Incubate with the appropriate concentration of primary antibody (first layer) 35 minutes in a dark and wet chamber at 37°C. Antibodies or Avidin are diluted to a working concentration in blocking solution;

7. Wash 3x3 minutes in 4xSSC/0.2%Tween, shaking;

8. Incubate with the appropriate concentration of secondary antibody (second layer) for 35 minutes in a dark and wet chamber at 37°C;

9. Wash 3x3 minutes in 4xSSC/0.2%Tween, shaking;

10. Optional third, forth layer of antibodies, washing in between 3x3 minutes in 4xSSCT, shaking;

11. Stain DNA with DAPI, 0.01-0.02g/ml 4xSSC/0.2%Tween, for 3 minutes;

12. Wash briefly in 4xSSC/0.2%Tween;

13. Mount hybridized areas in antifade (Vectashield);

14. Seal coverslips with colourless nail polish (see note 19 and comment 7).

Below one detection scheme is shown as an example for the detection of five and six different fluorochromes respectively that worked well in our hands

*In case of 5 colours we recommend to take out Alexa514.

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