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If the two primary antibodies are from different isotypes (such as mouse IgG1 and IgG2a), there may be isotype-specific secondary antibodies to choose from. The other option is to make direct conjugates of the primary antibodies through the use of antibody label kits, reactive dyes, or custom conjugations.
For blocking cells or tissues you may use 2–5% bovine serum albumin (fraction V, defatted BSA) or 5–10% normal serum of the species matching the host species of the secondary antibody. Other options include a mixture of BSA and serum or other purified proteins. We offer a ready-made blocking solution, BlockAid™ Blocking Solution (Cat. No. B10710), which is not species-specific. For cell or tissue samples, avoid the use of non-fat dry milk as a blocking agent as it contains a high level of phosphoproteins, histones, and biotin.
Aldehyde-based fixatives (e.g., formaldehyde, glutaraldehyde) crosslink various cellular components, which helps retain proteins and cell morphology, but some antigens can be masked by the crosslinking, requiring antigen retrieval methods to unmask the antigen binding sites. Also, aldehyde-based fixation requires permeabilization to allow entry of antibodies. Organic solvents such as methanol and acetone condense proteins and permeabilize the cells, but cell morphology can be compromised. Surface antigens can be labeled without fixation.
An optimal concentration may be between 1–10 µg/mL for cell and tissue labeling for microscopy, or 0.2–5 µg/mL for flow cytometry. A range of concentrations should be tested to determine what is optimal.
A common method for amplifying antibody detection is biotin-streptavidin detection, where a biotinylated secondary antibody is combined with subsequent labeling with a dye-conjugated streptavidin. This will amplify the signal by approximately 2–8 times, but endogenous biotin must be blocked beforehand. Another option is to use tyramide-signal amplification, where a horseradish peroxidase conjugate is used with a dye-labeled tyramide. This will amplify the signal by approximately 10–20 times, but endogenous peroxidase will need to be blocked. A final option may be to use a Qdot™ nanoparticle antibody or streptavidin conjugate, which can yield a signal as much as 40 times higher than a standard organic dye conjugate, depending on the Qdot™ color.
DAPI is a very common blue-fluorescent dye for nuclear counterstaining and gives very bright labeling on nuclei in fixed and permeabilized cells and tissues. However, it is considered to be a semi-permeant to impermeant stain and provides inconsistent staining of live cells. Hoechst™ 33342 dye is cell-permeant and stains with the same binding mechanism and fluorescent color; it is preferred for live-cell imaging and is just as good as DAPI for fixed cell labeling.
For live-cell imaging, the CellMask™ Plasma Membrane Stains are the most uniform and the slowest to be endocytosed. However, they are not the best choice if you wish to fix and permeabilize your cells, such as for antibody labeling. Wheat germ agglutinin (WGA) conjugates are also able to label live cells, or can label already formaldehyde-fixed cells. They can survive subsequent permeabilization with detergents, such as Triton™ X-100. If cells are already permeabilized, though, WGA will label internal structures as well. Thus, only an antibody against a plasma membrane protein can be used if cells are already permeabilized. Lipophilic cyanine dyes, such as DiI, will label all cell membranes in live cells, not just plasma membranes. This page will help you choose.
First, make sure your cells are maintained under conditions conducive to their viability during the staining and wash steps, including optimal temperature, CO2 percentage, and media/buffer. Not all reagents and protocols allow for this. Next, optimize your dye concentration and staining time. You want these to be high enough to give a strong signal over background, but the higher the concentration and the longer the staining time, the greater the chance that the reagent will have unintended effects on cell function and viability, as well as non-specific background labeling.
Some cell types accumulate phenol red, and this can pose a problem in the use of many fluorescent probes. Phenol red can quench visible-wavelength dyes and, although phenol red is non-fluorescent, various impurities may be fluorescent. We have many phenol red-free media to choose from. Our Live Cell Imaging Solution (HEPES-based) and our FluoroBrite™ DMEM have been optimized to be phenol red-free as well as to be non-autofluorescent.
Serum is necessary for general cell health and function, and some cell types are sensitive to its absence. Unfortunately, many dyes can bind to various components in serum limiting the uptake of dye into cells. Also, some serum may exhibit esterase activity which can cleave acetylated dyes (e.g., calcein, AM, FDA, etc.), preventing passive diffusion into live cells. As a general recommendation, if your cells of interest can tolerate a brief absence of serum during the stain incubation time, staining in serum-free medium or buffer is the best option. Cells may be returned to serum-containing medium after staining. If cells cannot tolerate any period without serum, you may need to increase the dye concentration to compensate for non-specific binding of dye to serum components. For acetylated dyes, try to use heat-treated serum or test the serum for esterase activity.
Most media contain phenol red, which can quench fluorescent dyes in the visible wavelengths. Most media also contain autofluorescent components, such as riboflavin, which can reduce signal-to-background. We offer FluoroBrite™ DMEM and HEPES-based Live Cell Imaging Solution, which have been optimized for fluorescent imaging. We also offer a number of media without phenol red. But if none of these are reasonable options for your experiment, then we also offer BackDrop™ Background Suppressor ReadyProbes™ Reagent, which can be added to quench media autofluorescence.
As dyes are illuminated for imaging, they will fade, or “photobleach”, leading to unwanted dimming and lower detection efficiency over time. An antifade mounting medium can greatly reduce photobleaching. If you wish to label live cells, use of ProLong™ Live Antifade Reagent is helpful. If you wish to mount fixed cells after labeling, and then image immediately and then discard, SlowFade™ Diamond Antifade Mountant stays liquid but has good refractive index. If you wish to mount your sample and then archive the slides, ProLong™ Diamond Antifade Mountant will harden to a better refractive index and allow for archiving of the sample for up to several weeks, or even months. Unlike other antifade mounting media, these work well with fluorescent proteins for immediate viewing (archiving fluorescent proteins is not possible), and they are packaged with or without DAPI. More information on these can be found here.
First, you need to determine what filter sets, light cubes, or laser wavelengths your imaging system is equipped with, as these will define what wavelength options to choose from. Often, the filter sets are named after dyes that fall in those wavelengths (for instance, a “FITC” filter set will work with fluorescein, Alexa Fluor™ 488, GFP, and most other “green” dyes). Then you should look at the dye options within those wavelengths and the spectra for those dyes, to see how well those dyes overlap with your filter specifications. Finally, you want dyes to be as spectrally separated from each other as possible to reduce the possibility of bleedthrough between filter sets. Often, dyes will differ in other characteristics. For instance, Alexa Fluor™ 488 dye is more photostable and less pH-sensitive than fluorescein. Our online SpectraViewer tool can be helpful to compare spectra of multiple dyes and match them with your filter or laser line characteristics.
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