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Selecting the right antibody is crucial for specific and sensitive experiments. In this chapter, you will learn about immunoglobulin class, species reactivity, and conjugate type so you can easily choose the right antibody for your cell staining.
Learn the differences and advantages of theses two immunofluorescence methods, and get the most of your antibodies in your experiments.
Learn about immunoglobulin class, species reactivity, and conjugate type so you can easily choose the right secondary antibody for your experiments.
Further information on this topic
What are pre-adsorbed secondary antibodies?
Pre-adsorption (also referred to as cross-adsorption) is an extra purification step introduced to increase the specificity of an antibody.
Learn about F(ab) fragment antibodies
What are they and what are the advantages of using them in immunohistochemistry?
Learn the difference between different antibody conjugates and how to choose the most appropriate for your application.
Knowing more about the fluorophores in your experiment is particularly important for multicolor experiments. In this section you will find out about the spectrum, emission, and absorption of your fluorophore.
Watch the mini video below to learn more about fluorophores.
Get the fluorochrome chart and guide so you can easily plan your multicolor fluorescent imaging experiments.
Learn all about the ICC and IHC protocol:
So there are many fluorochromes available. Each one of those will have it's own unique spectra for absorption and emission.
I've got a couple of examples here for PE and for Cy5.
The excitation wavelength is the signature wavelength at which the molecule is excited. So this is the wavelength at which you shine the laser.
The emission wavelength is the signature wavelength at which that fluorochrome emits a photon. So that's the wavelength at which you will detect.
So how does that work? So if you have a laser to that specific excitation length, it will hit an electron that's in resting state within the fluorochrome.
That electron absorbs the energy from that photon and jumps up to a higher energy level state.
It can't keep up that energy level state and eventually and quite quickly, that electron will loose the energy and drop back down to it's resting state.
In doing that, it releases any excess energy in the form of a photon
And that is fluorescence and that is what you're detecting.