I’ve had this trouble for a while and couldn’t figure out why. Typically I see people do in situ on slides, but I work with 96-well plates. I use the Perkin Elmer TSA plus kit (cy3/Fluorescein) for two colors. Two mRNA probes (one is Digoxigenin-labeled the other is Fluorescein labeled) were added to the sample and hybridized at 65C overnight. I use anti-Digoxigenin-POD for the first antibody reaction, coupling it with cy3 (red) for the TSA reaction. Then I use PH2.2 Glycine + tween20 to disable the first AB and wash many times. Then I add the second AB, anti-Fluorescein-POD, and couple it with fluorescein TSA….No matter how I optimized the conditions, the second color (green) showed up everywhere/non-specifically….any one had this problem before?????
Some cells autofluoresce, typically in the green. Look at the cells before the in situ treatment using your green cube and see if the green signal is already present. If it is, you will probably need to use a blue-emitting and red-emitting fluor and cube sets that exclude the green band.
Thanks for the reply. I looked at whole embryos and the auto-fluorescence was so weak that it wouldn’t even be picked up by our most sensitive cameras. I also suspected the endogenous peroxidase activity but that did not seem to factor in either since the anti-DIG + cy3 always worked. I thought of trying cy5 but we do not seem to have the filter for that wavelength…so i m in quite a pickle right now…
Problem seems to be solved. Not sure which step went wrong but the second signal began to appear when I diluted the second antibody 2X more and used less incubation time for the TSA reaction (about 3 min).