My goal was to distinguish between two forms of the same transcript, which are expressed in different cells, in midbrain cryosections. I designed the probes on the common region and on the truncated region. (LNA probes with double DIG labeling from Exiqon)
To detect the signal I used the Roche anti-DIG antibody coupled to the HNPP detection system (Roche), which uses of the Fast Red dye. I obtained completely red slices, even with the scramble probe where I should not see anything. The fact that I saw the slice completely red in the scramble makes me think that the problem is at the development stage, or that the anti-Dig antibody has been bound in a nonspecific manner everywhere. Has anyone some suggestions on how I can clean the signal? The high background could also be due to the concentration of probes that I used (25 or 50 nM) or it has to be excluded?
We use DD-ISH for Her2 from Roche in clinical histolab. Sometime I see red background, but well distinguishable from Centromer-signal. I think it is due to drying of the slide during long incubation or due to slightly overdigestion.
Do you use the Ultra stainer from Ventana-Roche for staining? with the prepared procedure for ISH? The DD-ISH procedure has some turning knobs for the red chromogen. eg. incubation time of anti-DIG ab, incubation time of red chromogen. Maybe you have similar possibilities.
The mounting medium is quite critical for fast red because the red can diffuse out all over the section. Sections thicker than @10um will also cause this problem
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