Actin Staining in Fixed Cells
In fixed cells, actin structures can be visualized by fluorescent phalloidins (2, see protocol below), actin antibodies (1, see protocol below), or electron microscopy (3). Correctly designed fluorescent phalloidins only binding to the native quaternary structure of F-actin and therefore have a low background. To create the correct fixation conditions for phalloidin binding paraformaldehyde must be used as the fixative because it retains the quanternary protein structure which is necessary for high affinity. Methanol destroys the native conformation and hence is not suitable for actin staining with phalloidin. Antibodies recognize both monomer and polymer (filamentous or F-actin) actin and hence tend to have a high background compared to probes that bind only F-actin, but are compatible with methanol fixation and may be useful when co-staining antibodies requiring methanol fixation.
Utilizing Fluorescent Phalloidins
Fluorescent phalloidins vary greatly in their staining characteristics because of their structure, for example a positively or negatively charged fluorescent dye will create higher backgrounds by binding non-specifically through ionic interactions with other proteins. The Acti-stain series of dyes are non-ionic and therefore create very low backgrounds, they can even be used without washing steps after the staining procedure. Another aspect for low background fluorescence and increased brightness is the amount of quenching that is caused by the close proximity of the dye to phalloidin (4) or dye to actin (5). We have screened many combinations of dye and linker and created the optimal combinations which allows brightness to remain as high as the original fluorescent dye.
Acti-stain Dye Properties
The brightness of fluorescent probes is controlled by environmental factors, in addition to the quenching aspect mentioned above, there is also a rotational consideration, if the dye is allowed to move freely there is a reduction of emitted light, rather the energy is dissipated as heat. The rotational status of dyes judged by their quantum yield allows the development of brighter dyes. The brightness of fluorescent phalloidins is also affected by their affinity for F-actin. Unlabeled phalloidin has a Kd of approx. 36 nM, whereas fluorescent conjugates of phalloidin can vary from 50 nM to 20 µM (Ref. 4 and Figure 2 below for Acti-stain 488), the lower the Kd the higher the affinity and hence the better binding is achieved which results in a brighter stain. Acti-stain dyes have Kd's in the lowest range 50 to 100 nM which creates bright fluorescence signals (for more information click here).
Fluorescent dyes can be photo-deactivated by intense microscope light sources. This affect can be reduced with anti-fade solutions such as DABCO (see BK005 kit below), and also by engineering the dye to remove ionizable protons. Many of the modern dyes, Acti-stain ones included, have replaced ionizable protons with fluorine which does not ionize as readily and therefore does not bleach as much as traditional dyes such as fluorescein (6). Fluorescent phalloidins have two characters which affect signal stability, these are photo-bleaching as described above and their affinity for F-actin. The latter aspect can be observed with low affinity conjugates such as coumarin phalloidin (4) which are released from F-actin during storage before microscopic observation.
Yeast cells contain highly divergent actin proteins but these still bind to some fluorescent phalloidins such as Acti-stain 488 as shown adjacent.
For the performance and chemical characteristics of the Acti-stain dyes click here.
Utilizing Actin Antibodies For Immunofluorescence
Anti Pan-Actin Antibodies can also be utilized for immunfluorescent staining of cells to visualize actin. Importantly, fixation and handling of the samples is critical for effective pan-actin antibody staining. Cytoskeleton's actin antibody is compatible with methanol fixation (as shown in the figure), and may be useful when co-staining antibodies require methanol fixation. For a detailed protocol click here.
Comparison of fixation strategies used for immunofluorescence images of mouse swiss 3T3 cells stained with anti-actin antibody
Swiss 3T3 fibroblasts were plated on glass coverslips, grown to 30% confluency in DMEM plus 10% FBS, and serum starved for 24 h in media containing 1% FBS followed by 24 h in serum free media. Cells were treated with a buffer control or 1mg/ml of the Rho Activator (CN03) for 2 h at 37C.5% CO2 to induce stress fibers. The 3T3 cells were then fixed with the followin methods: 1. PFA / phalloidin stainng, 2. PFA-MeOH / AAN02 (actin antibody) staining, 3. MeOH / AAN02 staining, 4. PFA / AAN02 staining. For condition 2, PFA fixation was followed by permeabilization with methanol (see protocol below for more details). Immunofluorescence staining using a 1:500 dilution of anti-actin antibody is shown (green). The primary antibody was detected with a 1:500 dilution of goat anti-mouse Alexa Fluor 488 conjugated antibody. Images were taken with a 40X objective lens.
Cytoskeleton's actin staining products for fixed cells:
Acti-stain™ 488 (very stable green fluorescence, fixed cell stain)
Acti-stain™ 535 (red fluorescence, fixed cell stain)
Acti-stain™ 555 (very stable red fluorescence, fixed cell stain)
Acti-stain™ 670 (far-red fluorescence, fixed cell stain)
F-actin Visualization Biochem Kit™ (rhodamine)
Anti Pan-Actin antibody (#AAN02) (Mouse Monoclonal Antibody)
In living cells actin structures can be observed by incorporating fluorescently labeled actin, expressing GFP-actin, or a fluorescently labeled actin binding protein sub-domain. Fluorescent actin is the most accurate reporter of actin structures but it is sometimes difficult to load into cells which is accomplished by microinjection or a protein transfection reagent (see protocol below). Recently, cell-permeable and non-cytotoxic live cell imaging probes (SiR-Actin and SiR-Tubulin) were introduced in a landmark paper published in Nature Methods. To learn more about these live cell imaging probes including protocols, videos, and images click here.
GFP-actin DNA vectors are readily transfected into cells, however some cells e.g. primary cells, are recalcitrant to transfection technology but this will undoubtedly improve over time with the introduction of new vectors such as viral particles. GFP-actin is also known to interfere with normal cytoskeletal dynamics producing aberrations in nuclei number per cell and myosin processivity (7). F-actin binding protein tags are useful conjugates to probe F-actin in live cells (8,9) but they also need the bulky GFP reporter to produce a signal.
SiR-Actin applied to tissue or cells in culture will transition the membrane and label F-actin structures. Imaging can proceed after one or two hours.
Rhodamine non-muscle actin (Cat. # APHR) injected into CHO cells. The labeled actin rapidly incoporates into the cellular actin cytoskeleton and allows real time observation of actin dynamics.
This section contains protocols for the following applications:
There are several methods that are used for fluorescent staining of actin filaments in tissue culture cells. The fixation procedure is critical for obtaining faithful representation of the F-actin distribution within the cell. The fixation method should be selected on the basis of the experimental requirements. Fixing tissue culture cells in paraformaldehyde or glutaraldehyde results in excellent actin filament staining and good lamellipodia preservation.
Stabilized fluorescent actin filaments are an excellent substrate for in vitro actin motility assays used in the study of myosin motor proteins (2). Acti-stain™ 488 phalloidin binding has no effect on actin activation of myosin ATPase in vitro.
Cells or tissue slices are grown in 3D collagen matrices to more accurately replicate the in vivo cellular milieu that cells function in to study how cell morphology and function respond to the extracellular environment confining the cell. To faithfully capture the integrity and structure of the F-actin cytoskeleton (and any changes it undergoes) with a fluorescent phalloidin stain, proper fixation and handling of the cells/slices is necessary. For fixation of cells for phalloidin staining, methanol must be avoided and instead, paraformaldehyde or glutaraldehyde should be used as they provide excellent actin filament staining and good lamellipodia preservation.
Fluorescent Phalloidin Staining Protocol
Phalloidin Staining Protocol
For effective staining with anti pan-actin antibodies proper fixation and handling of the cells/slices is necessary (see above for example). For fixation of cells with anti pan-actin antibody, we recommend the following method which utilizes sequential paraformaldehyde fixation and methanol fixation/permeabilization followed by Triton X-100 permeabilization.
IF Method (PFA/Methanol/Triton X-100 Fixation of Swiss 3T3 cells):
1. Lazarides E, and Weber K. 1974. Actin antibody: The specific visualization of actin filaments in non-muscle cells. PNAS USA 71, 2268-2272.
2. Wulf F, Deboben A, Bautz FA, Fualstich H, and Wieland TH. 1979. Fluorescent phallotoxin, a tool for the visualization of cellular actin. PNAS USA71, 4498-4502.
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10) Oda T, Namba K, and Maeda Y. (2005). Position and Orientation of Phalloidin in F-Actin Determined by X-Ray Fiber Diffraction Analysis. Biophys. J, 88, 2727–2736.