Abstract:
The cytoskeleton is mainly composed of microfilaments, microtubules, and intermediate filaments. Microfilaments are primarily made of actin, existing in globular monomeric G-actin and filamentous F-actin forms. Assembly of microfilaments involves polymerization of G-actin into F-actin. Phalloidin, a cyclic peptide isolated from Amanita phalloides, binds F-actin with high specificity and affinity, and thus is widely used for staining cellular microfilaments.
Choosing the right phalloidin product, correct fixation and permeabilization, and appropriate working concentration and incubation conditions are critical for successful staining. Frequently encountered failure points when using phalloidin to stain cytoskeleton are addressed below.
Q1: Can samples be used without fixation?
No. Fluorescent phalloidin conjugates cannot cross the cell membrane due to size and chemical properties, making fixation and permeabilization mandatory.
Q2: Which fixative should be used?
For F-actin staining, paraformaldehyde (PFA) is essential—it preserves quaternary structure, unlike methanol which disrupts protein conformation. Recommended: 4% PFA, room temperature for 10 min. Glutaraldehyde fixation is also acceptable. Among tested fixatives (e.g., in H1299 cells), 4% PFA yielded the clearest microfilament structure with TRITC-Phalloidin.
Q3: Is permeabilization required?
Yes. After fixation, permeabilization is crucial. Recommended: 0.1% Triton X100 at room temperature for 5 min.
Q4: What about working concentration and incubation time?
Depends on:
Type of fluorophore conjugate and stock concentration. Unlabeled phalloidin has Kd ≈36 nM; labeled versions vary between 50 nM–20 µM. Know your stock concentration.
Cell type. Typically 80–200 nM is used; 100 nM is common. Certain cells (e.g., platelets, dental pulp stem cells, osteoclasts) may need 5–10 µM.
Default incubation: 30 min. You can adjust or incubate overnight at 4 °C for low signal.
Q5: How to choose among fluorophore conjugates?
FITC- and TRITC-phalloidin are reliable classics.
For multiplexing, choose dyes that don’t overlap with other antibodies.
For high brightness and photostability, consider Alexa Fluor or iFluor series.
Q6: Can phalloidin be used on tissue sections?
Yes—with caveats. Paraffin embedding solvents and wax can hinder binding; even after deparaffinization, structural changes often reduce staining quality. Tissue staining ranking: cells > frozen sections > paraffin.
Q7: Is morphological variability normal?
Yes. F-actin arrangement changes with cell cycle. For example, mitotic Swiss 3T3 cells show different F-actin morphology compared to interphase cells.
Q8: What is the recommended staining sequence when also staining proteins or nuclei (e.g., with DAPI)?
After fixation and permeabilization, first perform immunostaining for target proteins, then stain microfilaments with fluorescent phalloidin, and finally stain nuclei with DAPI. DAPI and phalloidin can be co-incubated in PBS without interference.
Using proper reagents and methods greatly enhances staining efficiency. Wishing everyone beautiful cytoskeleton images!
References:
Luo Guo, Bao Yuxin, Li Jin. Influence of different fixatives on cytoskeleton fluorescence staining [J]. Journal of Third Military Medical University, 2011, 33(7):753755.
Anderson MT, Sherrard K, Horne-Badovinac S. Optimized Fixation and Phalloidin Staining of Basally Localized F-Actin Networks in Collectively Migrating Follicle Cells. In: Giedt MS, Tootle TL, eds. Drosophila Oogenesis. Methods in Molecular Biology, vol 2626. Humana, New York, NY, 2023.
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