Actin Staining Protocols

Protocol 1 - Fixing and staining tissue culture cells with fluorescent phalloidin (F-actin), Dapi (DNA) and an antibody.

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.

Reagents

1. Acti-stain™ 488 Phalloidin (Cat. # PHDG1).
2. Semi-confluent Swiss 3T3 cells grown on 25 x 25 mm glass coverslips grown in DMEM plus 10% fetal bovine serum and 1% Fungizone.
3. Either obtain the F-actin staining kit from Cytoskeleton, Inc. (Cat. # BK005) or prepare Reagents 4 thru 8 below.
4. Phosphate-buffered saline (PBS, 50 mM potassium phosphate pH 7.4, 50 mM NaCl).
5. Fixative solution (3.7% paraformaldehyde in PBS, pH to 7.0 is necessary).
6. Permeabilization buffer (0.5 % Triton X-100 in PBS).
7. Antifade mounting medium (Fluka BioChemika, Cat. # 10981).
8. Bovine serum albumin solution in PBS (prepared fresh, 10 mg/ml BSA in PBS)
9. Primary antibody at 1 : 100 dilution in PBS plus 1 mg/ml BSA (prepared fresh)
10. Secondary antibody (rhodamine conjugate) at 1:250 dilution in PBS plus 1 mg/ml BSA (prepared fresh)
11. 100 nM DAPI (4' 6-diamidino-2-phenylindole) in PBS.
12. Glass microscope slide (25 x 75 x 1 mm).
13. Coverslip sealing solution (clear nail polish).

Equipment

1. Fluorescent microscope with FITC excitation filter at 450-480 +/- 20 nm and emission filter at 535 +/- 20 nm for Acti-stain™ 488, and an excitation filter at 355 +/- 20 nm and emission filter at 440 +/- 20 nm for DAPI.
2. Digital CCD camera.

Method

1. Grow tissue culture cells on glass coverslips until semi-confluent.
2. Prepare 100 nM working stock of Acti-stain™ 488 phalloidin by diluting 3.5 μl of 14 μM labeled stock phalloidin into 500 μl of PBS. Keep at room temperature in the dark.
3. Remove culture media and gently wash the cells once with PBS at room temperature (RT).
4. Move the coverslip to a piece of parafilm in a humid chamber and add 200 µl of fixative solution to each coverslip for 10 min at RT.
5. Wash the cells once with PBS at RT for 30 s.
6. Permeabilize the cells by pipetting 200 µl of permeabilization buffer on to the coverslip and incubating for 5 min at RT.
7. Wash the cells twice with PBS at RT for 30 s.
8. Incubate with 1 mg/ml BSA in PBS for 1h at RT. Note: Italicized sections can be removed if antibody staining is not required.
9. Wash once with PBS at RT for 30 s.
10. Pipette 200 µl of primary antibody solution and incubate at RT for 1h (or 2h if dense cellular location such as nucleus).
11. Wash three times with PBST at RT for 5 min each wash.
12. Pipette 200 µl of secondary antibody solution and incubate at RT, in the dark, for 1h (or 2h if dense cellular location such as nucleus)
13. Wash three times with PBS at RT for 5 min each wash.
14. Pipette 200 μl of 100 nM Acti-stain™ 488 phalloidin plus 100 nM Dapi solution in PBS. Incubate at RT in the dark for 30 min.
15. Wash the coverslip three times in PBS for for 30 s each wash.
16. Remove liquid from the coverslip by touching the corner on a paper towel and invert on a drop of antifade mounting media on a glass slide.
17. Leave for 20 min to dry and seal each side with nail polish.
18. Either view immediately of Store the slides in the dark at 4°C for upto 24 h.
19. Typical F-actin staining results are shown in Figure 3 (above).

Protocol 2 - Labeling F-actin in vitro for microscopic molecular studies.

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.

Reagents

1. Actin protein (250 μg, Cat. # AKL99-A).
2. General Actin Buffer (5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2; Cat. # BSA01).
3. 10x Polymerization Buffer (500 mM KCl, 20 mM MgCl2, 10 mM ATP; Cat. # BSA02).
4. Acti-stain™ 488 Phalloidin (Cat. # PHDG1).

Equipment

1. Fluorescent microscope with FITC excitation filter at 450-480 +/- 20 nm and emission filter at 535 +/- 20 nm for Acti-stain™ 488, and an excitation filter at 355 +/- 20 nm and emission filter at 440 +/- 20 nm for DAPI.
2. Digital CCD camera.

Method

1. Resuspend rabbit muscle actin (Cat. # AKL99-A) to 1 mg/ml with 250 μl of General Actin Buffer supplemented with 0.2 mM ATP and 1.0 mM DTT. Mix well and leave on ice for 1 h.
2. Polymerize the actin with 1/10th the volume of Polymerization Buffer for 1 h at room temperature.
3. Dilute the polymerized actin filaments 100 fold in 1x Polymerization Buffer containing 70 nM Acti-stain™ 488 phalloidin (2.5 μl of 14 μM stock plus 500 ul of polymerization buffer).
4. Spot 1 μl of the labeled actin into a drop of anti-fade mounting media on a microscope slide.
5. Place a coverslip over the drop and remove excess liquid with a tissue.
6. Examine the fluorescent filaments by microscopy. Actin filaments will have an average length of 2-10 μm and are stable at 4°C in the dark for 1 week.

Protocol 3 - Microinjection of rhodamine muscle actin into tissue culture cells.

Reagents

1. Rhodamine muscle actin (Cat. # AR05).
2. General Actin Buffer (5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2; Cat. # BSA01).
3. Tissue culture cells seeded on glass coverslips.

Equipment

1. Microinjector apparatus.

Method

1. Resuspend rhodamine muscle actin to 2 mg/ml with General Actin Buffer supplemented with 0.2 mM ATP and 1 mM DTT. Leave on ice for 1 h to depolymerize actin oligomers. Note: If calcium ions affect the cells, rhodamine muscle actin can be resuspended in pure water.
2. Centrifuge the muscle actin solution at 14,000 x g at4°C for 10 min. (or better 100,000 x g at 4°C for 10 min). Remove the top 90% of the supernatant to a new tube and load the microinjection needle by capillary action.
3. Microinject rhodamine muscle actin into cells maintaining positive pressure in the needle to prevent blockage. Note: salt in the tissue culture media and in the cell cytoplasm can result in actin polymerization within the needle.
4. View cells in a temperature controlled microscope stage using low light and a CCD camera.

Protocol 4 - Fixing and staining tissue culture cells or slices grown in a 3D collagen matrix with fluorescent phalloidin

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

Reagents

1. Semi-confluent 3D tissue culture cells or tissue slices (see supplemental details below for preparing these cultures)
2. Acti-stain 488 phalloidin (Cat. # PHDG1). Other options include Acti-stain 535 (Cat. # PHDR1), Acti-stain 555 (Cat. # PHDH1), and Acti-stain 670 (Cat. # PHDN1)
3. Either obtain the F-actin staining kit from Cytoskeleton, Inc. (Cat. # BK005) or prepare reagents below
4. Fixative solution (3.7% paraformaldehyde in PBS, pH to 7.0 is necessary)
5. Permeabilization buffer (0.5 % Triton X-100 in PBS)
6. Antifade mounting medium (Fluka BioChemika, Cat. # 10981)
7. Coverslip sealing solution (clear nail polish)

Equipment

1. Fluorescent microscope with FITC excitation filter at 450-480 +/- 20 nm and emission filter at 535 +/- 20 nm for Acti-stain™ 488. For Acti-stain red fluorophores, the excitation filter is 535 +/- 20 nm and emission filter at 585 +/- 20 nm for Acti-stain 535 and 555. For Acti-stain 670, the excitation filter is at 630 +/- 20 nm and the emission filter is at 680 +/- 20 nm.
2. Digital CCD camera.

Method

Phalloidin Staining Protocol

1. Reconstitute phalloidin according to manufacturer’s directions. Dilute to working concentration as directed in PBS. Keep working stock of fluorescent phalloidin in the dark at room temperature.
2. Fix cells in the collagen gels with 3.7% (v/v) paraformaldehyde 10 min at room temperature.
3. Rinse 3 times in PBS.
4. Permeabilize cells with 0.1 -0.5% (v/v) Triton X-100 for 10 min at room temperature.
5. Rinse 3 times in PBS.
6. Cut collagen gels into ~1 mm³ samples.
7. Stain with 0.165 μM Acti-stain phalloidin for 30 min in the dark at room temperature. Note: This concentration may need to be empirically determined. Cell nuclei can be stained with 100 nM DAPI in PBS in concert with phalloidin if desired.
8. Wash 3 times with PBS.
9. Place stained samples on glass slides and gently press with a glass coverslip using a drop of anti-fade mounting medium to prevent photo-bleaching.
10. If possible, seal each side of coverslip with nail polish.
11. Either view immediately or store the slides in the dark at 4°C for up to 24 hrs.
12. Visualize the cells in stained samples with an appropriate fluorescence microscope set-up. For example, a confocal fluorescent microscope with a 63X oil-immersion lenses at the appropriate filter excitation and emission settings.
13. For analysis of staining in 3D gels, collection of 5-10 representative maximal projection z-stacks from each sample are useful to determine the average brightness of cells within each image.

Protocol 5 - Fixing and staining tissue culture cells with anti-pan actin antibody

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.

Reagents

1. Semi-confluent tissue culture cells
2. Anti pan-actin antibody (Cat. # AAN02).
3. Ice cold methanol
4. Fixative solution (3.7% paraformaldehyde in PBS, pH to 7.0 is necessary)
5. Permeabilization buffer (0.5 % Triton X-100 in PBS)
6. Antifade mounting medium (Fluka BioChemika, Cat. # 10981)
7. Coverslip sealing solution (clear nail polish)

Equipment

1. Fluorescent microscope with the proper filters compatible with your secondary of choice: Method uses FITC excitation filter at 450-480 +/- 20 nm and emission filter at 535 +/- 20 nm for Alexafluor™ 488.
2. Digital CCD camera.

Method

IF Method (PFA/Methanol/Triton X-100 Fixation of Swiss 3T3 cells):

1. 3T3 cells were plated on poly-lysine and laminin coated coverslips and grown to appropriate densities (~ 25% confluency).
2. Remove culture media and wash cells once with 1x PBS.
3. Fix cells for 10 min at room temperature (RT) with 3.7% PFA (paraformaldehyde in 1X PBS) (fresh, methanol free).
4. Wash cells two times with 1x PBS.
5. Permeabilize cells with 100% Methanol 4^o C for 30 min.
6. Wash cells two times with 1x PBS.
7. Permeabilize cells with 0.5% Triton X-100 RT for 15 min.
8. Wash cells two times with 1x PBS.
9. Block cells with 1% BSA, 0.3 M glycine in 1x PBS + 0.01% Tween (PBST) for 60 min RT.
10. Incubate cells with 0.2 mg/ml AAN02 in 1x PBS for 90 min at RT.
11. Wash cells once with 0.1% Triton-X-100 in 1x PBS.
12. Wash cells three times with PBST.
13. Incubate cells with (Alexa Fluor® 488) secondary antibody 1:500 in PBST for 60 min at RT (minimize light exposure).
14. Wash cells three times with 1x PBS.
15. Incubate cells with DAPI stain 1:500 in 1x PBS for 20 min at RT.
16. Wash cells three times with 1x PBS.
17. Dip coverslips in water.
18. Mount on glass slide with mounting media, and sit for 20 min.
19. Seal edges with nail polish for 10-20 min.

Related Products

• Rhodamine Phalloidin (red fluorescence fixed cell stain)
• F-actin Visualization Biochem Kit™ (rhodamine)
• Pan-Actin Antibody (Mouse Monoclonal)
• All Actin Products page
• Activation Assays
• G-LISA Small G-protein Activation Assays