I have not used this specific kit, but I have worked with OPAL tyramides and other tyramides to generate multispectral images. This is not trivial, requires either a sophisticated widefield fluorescence microscope with plenty of filter cubes and probably quite expensive software. If you have a confocal microscope, you need plenty of lasers or a white laser and a 405nm laser, perhaps more.

The kit seems to come with three antibodies and you have to find 3 more that go along with the ones that are in the kit. Even if you have 6 good antibodies, you need to figure out the sequence of antibody application, the concentrations, and which OPAL reagent or other tyramide-fluorescent dye to use at each step. With 7 fluorescent dyes this is challenging.

I managed to get 7 colors on one section but it is getting to the limits of the confocal microscope I use. You have to deal with bleed-through; for some highly expressed antigens it is difficult to eliminate all antibodies in the microwave stripping step etc etc. Currently, I have settled for 6 color stains (5 antibodies and one DNA dye) with a set of 4 tyramides from PerkinElmer, Fisher, and Biotium, one regular secondary directly conjugated to a fluorescent dye and a DNA dye called BOBO1.

I think PerkinElmer will probably go for more colors but translating this to individual labs will be difficult. At that level, you propbably have to buy their Vectra system or a confocal microscope with a lot of lasers, you probably need something like a white laser like in a Leica SP8 X confocal. I have not tried that either of them.

Bottom line: this is challenging and requires a lot of testing and trial-and-error. Tyramides are relatively expensive (you make up some of the cost by significantly reducing primary antibody concentration and background reduction due to lower antibody concentrations).

My advice: carefully select the three additional antigens you want to include, make sure your microscope has the capability to separate the dyes well, re-determine the antibody concentrations you have to use with the tyramide system (rough rule of thumb: around 3- to 10-fold less than with conventional secondaries). Check which dyes will have the most bleed through and chose the antigens accordingly to be able to take care of this problem (e.g. if you put two nuclear antigens in these "channels" that tend to have bleed through, it may be hard to distinguish the signal). I am sure the PerkinElmer Vectra system has plenty of detailed and sophisticated features to control for and eliminate problems with this type of bleed-through; and most confocal microscopes have software features that can be used to approach this problem.

Some antigens (in my experience less than 10% of the antigens) do not like the repeated cooking. This can look ugly or just result in plain failure to result in any signal. These antigens would have to go at the beginning of the procedure or have to be excluded...

Good luck.

I have not used this specific kit, but I have worked with OPAL tyramides and other tyramides to generate multispectral images. This is not trivial, requires either a sophisticated widefield fluorescence microscope with plenty of filter cubes and probably quite expensive software. If you have a confocal microscope, you need plenty of lasers or a white laser and a 405nm laser, perhaps more.

The kit seems to come with three antibodies and you have to find 3 more that go along with the ones that are in the kit. Even if you have 6 good antibodies, you need to figure out the sequence of antibody application, the concentrations, and which OPAL reagent or other tyramide-fluorescent dye to use at each step. With 7 fluorescent dyes this is challenging.

I managed to get 7 colors on one section but it is getting to the limits of the confocal microscope I use. You have to deal with bleed-through; for some highly expressed antigens it is difficult to eliminate all antibodies in the microwave stripping step etc etc. Currently, I have settled for 6 color stains (5 antibodies and one DNA dye) with a set of 4 tyramides from PerkinElmer, Fisher, and Biotium, one regular secondary directly conjugated to a fluorescent dye and a DNA dye called BOBO1.

I think PerkinElmer will probably go for more colors but translating this to individual labs will be difficult. At that level, you propbably have to buy their Vectra system or a confocal microscope with a lot of lasers, you probably need something like a white laser like in a Leica SP8 X confocal. I have not tried that either of them.

Bottom line: this is challenging and requires a lot of testing and trial-and-error. Tyramides are relatively expensive (you make up some of the cost by significantly reducing primary antibody concentration and background reduction due to lower antibody concentrations).

My advice: carefully select the three additional antigens you want to include, make sure your microscope has the capability to separate the dyes well, re-determine the antibody concentrations you have to use with the tyramide system (rough rule of thumb: around 3- to 10-fold less than with conventional secondaries). Check which dyes will have the most bleed through and chose the antigens accordingly to be able to take care of this problem (e.g. if you put two nuclear antigens in these "channels" that tend to have bleed through, it may be hard to distinguish the signal). I am sure the PerkinElmer Vectra system has plenty of detailed and sophisticated features to control for and eliminate problems with this type of bleed-through; and most confocal microscopes have software features that can be used to approach this problem.

Some antigens (in my experience less than 10% of the antigens) do not like the repeated cooking. This can look ugly or just result in plain failure to result in any signal. These antigens would have to go at the beginning of the procedure or have to be excluded...

Good luck.

Hi Thomas;

Thanks, your advice is very helpful.