Selection of Fluorochromes for Multicolor ICC IF

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Fluorochromes Selection for Multicolor ICC/IF

Sample Preparation for ICC/IF Experiments

Fixation & Permeabilization

Blocking for ICC-IF Assay

Antibody Selection in ICC/IF

Detection Methods for ICC IF

Controls for ICC/IF Experiments

Counterstaining & Mounting

Useful Links

Multicolor ICC/IF Protocol

Troubleshooting ICC/IF

Secondary Antibodies

Organelle Markers Guide

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ICC Handbook

Rules for Selection of Fluorochromes

Designing a successful ICC/IF experiment requires the selection of suitable fluorochromes. Beginners often find it challenging to choose optimal fluorochrome combinations. Some tips are provided below to simplify the selection process and to minimize potential errors.

Fluorochrome and microscope compatibility Become familiar with the lasers and filter sets of your microscope to ensure you are selecting fluorochromes that are optimally excited and detected by them. Spectra viewers and fluorochrome reference charts are useful tools to determine the maximum excitation and emission wavelengths of the fluorochromes under consideration. Some filters such as FITC/TRITC indicate the fluorochrome to use in the filter name. In addition, a rough guide for laser/filter selection is provided by the number listed after some dyes, such as 488 in Alexa Fluor® 488 or DyLightTM 488.
Extinction coefficients (ε) and brightness of fluorochrome The brightness of fluorochromes is directly proportional to the extinction coefficient, a measurement of the fluorophore’s probability of absorbing a photon of light. For example, a dim fluorochrome, such as DyLightTM 350, has ε value = 15,000, whereas a brighter member of this family, DyLightTM 650, has an ε value = 250,000. In multi-color ICC/IF, it is advisable to associate antigen abundance with the brightness of a fluorochrome. In general, we recommend using the brightest fluorochrome to detect the least abundant antigen and to select the dimmest fluorochrome to detect the most abundant antigen.
Quantum yields (Φ) of fluorochrome Fluorescence quantum yield is a measurement of emission efficiency and is calculated by the formula: ΦF = number of emitted photons/number of absorbed photons. The maximum quantum yield possible (ΦF = 1) signifies a 100% efficient fluorescence process. An example of a fluorochrome with high quantum yield is Alexa Fluor® 488 which has ΦF= 0.92.
Photobleaching & pH compatibility Photobleaching refers to a catabolic photochemical reaction that results in a reduction in fluorescence intensity. Conventional fluorochromes such as FITC and PE are sensitive to photobleaching whereas fluorochromes including Alexa Fluor®, DyLightTM or HiLyteTM dye families are more photostable. If using FITC or PE in an experiment, lower the intensity or exposure time of the excitation light or use an anti-fade reagent containing mounting medium to reduce potential issues. The fluorescence intensity of some conventional fluorochromes, including FITC is highly sensitive to pH and alcohols, which limits their usefulness in protocols using acidic buffers and alcohol-containing reagents. In contrast, improved dyes such as Alexa Fluor®, DylightTM etc. are more tolerant to acidic buffers and alcohols.
Spectral overlap, the bleed-through effect Bleed-through or spill-over effect is the detection of fluorochromes in adjacent filter sets. This can make it difficult to distinguish interfering fluorescence from the fluorochrome of interest, which is particularly problematic for co-localization studies. For example, the most commonly used nuclear counterstain DAPI cannot be used in combination with blue emitting fluorochromes such as Alexa Fluor® 405, DyLightTM 405 or HiLyte 405. Therefore, it is critical to select fluorochromes with minimal spectral overlap for multi-color ICC/IF.