Immunocytochemistry/immunofluorescence (ICC/IF) involves visualization of antigens in cultured or smeared cells with the use of fluorochrome-labeled antibodies. Various combinations of fluorochrome-labeled antibodies make it possible to simultaneously detect multiple antigens in the same sample. However, every experiment involving multiplex or multicolor ICC/IF requires selection of an optimal set of fluorochromes. Poor selection of fluorochrome combinations can make it difficult to distinguish individual antigens, especially when the targets under consideration are co-localized at the sub-cellular level. Here we have outlined key rules for selecting fluorochromes to minimize potential errors and to simplify the selection process.
1. Check for fluorochrome(s) compatibility with your microscope’s filter sets
Use spectra viewers and fluorochrome reference charts to determine the maximum excitation (Ex) and emission (Em) wavelengths of the fluorochromes. Ensure that the fluorochromes under consideration are optimally excited and detected by the laser and filter sets of your microscope. Names of some filters (e.g. FITC/TRITC) or the numbers listed in some dyes (e.g. 488 in DyLight 488TM) can be used for determining the fluorochrome-microscope filter sets compatibility.
2. Associate brightness of fluorochromes with the relative expression of target antigens
Based on published reports or data from your lab records, categorize the antigens according to their relative expression in samples to be tested. Select the brightest fluorochrome for detecting the least abundant antigen and vice versa. The brightness of a fluorochrome is directly proportional to the extinction coefficient (ε), a measurement of the fluorophore’s probability of absorbing a photon of light. For example, DyLightTM 350 has an extinction coefficient value of 15,000 (dim) while DyLightTM 650 has an extinction coefficient of 250,000 (bright). Accordingly, DyLightTM 350 would be a good choice for detecting a highly expressed protein (e.g. beta actin) and DyLightTM 650 can be considered for a relatively low expressed protein (e.g. LC3) under basal conditions.
3. Do not select fluorochromes which are sensitive to photobleaching, alcohols or pH
Photobleaching is a catabolic photochemical reaction that results in a reduction in fluorescence intensity. Conventional fluorochromes (e.g. FITC and PE) are more sensitive to photobleaching effects while relatively modern dye families including Alexa Fluor® and DyLightTM are highly photostable. When options are limited, use an anti-fade reagent and lower the intensity or exposure time of the excitation light for reducing photobleaching effects. Similarly, Conventional fluorochromes are sensitive to pH and alcohols and are not suitable in protocols involving acidic buffers and alcohol-containing reagents.
4. Minimize the bleed-through effect by considering the spectral overlap of fluorescent molecules
It is very important to select fluorochromes and/or counterstaining dyes which have minimal to negligible spectral overlap. This will help reduce the bleed-through or spill-over effects, the detection of fluorochrome signal in adjacent filter sets. As an example, FITC cannot be used with Alexa Fluor® 488. If biological or fixative-induced autofluorescence is suspected in your samples, avoid fluorochromes which might show spectral overlap with emission spectra of autofluorescence generating molecules in your samples.
For additional tips on fluorochrome selection and a quick refresher on basics, protocols, and troubleshooting of multicolor ICC, read our Immunocytochemistry (ICC) Handbook.