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Introduction, Advantages, and Experimental Procedures for Alexa Fluor 488–Labeled Phalloidin
Phalloidin is a cyclic peptide isolated from the fungus Amanita dhalloides. It binds competitively to F‑actin. Fluorescently labeled phalloidin can selectively bind to F‑actin at the nanogram level and is readily soluble in water, providing a highly convenient labeling method for visualizing and quantifying actin in tissue sections, cultured cells, and cell-free systems. Moreover, Alexa Fluor 488–labeled phalloidin emits bright, stable green fluorescence, exhibits high specificity in staining, and offers excellent contrast, making it well suited for both qualitative and quantitative analysis of F‑actin.
Compared with actin antibodies, Alexa Fluor 488 phalloidin offers the following advantages:
1. It binds to actin subunits in a 1:1 ratio and does not bind to G-actin, yielding superior staining compared to actin antibodies.
2. Moreover, this product exhibits no species‑specific binding, making it widely applicable.
3. Non-specific signals labeled with phalloidin are negligible, resulting in good image contrast.
4. The dye is fully compatible with other fluorescent stains used for cell analysis, including fluorescent proteins, nanocrystals, and other Alexa Fluor conjugates (including Alexa Fluor–conjugated secondary antibodies). • F‑actin bound by this product retains many of the intrinsic biological properties of actin itself.
488 Phalloidin Staining Product Recommendations
[Brand] Heyuan Liji Bio
[Product] Live Tissue Cryopreservation and Revival Reagent
[Item Number] AC05L463
[Specification] 1 T
488 Phalloidin Staining Product Usage Instructions
Step 1: Prepare the 1× Alexa Fluor 488–labeled phalloidin working solution by pipetting 1 μL of the 1000× Alexa Fluor 488–labeled phalloidin stock solution into 1 mL of PBS buffer containing 1% BSA. This will yield a 1× working solution.
Step 2: Cell Staining Procedure
(1) Culture the cells overnight or longer until they reach a confluence of 50–60%.
(2) Aspirate the culture medium, then wash the cells twice with 1× PBS (pH 7.4) pre-warmed to 37°C.
(3) Fix the cells using a PBS solution containing 3–4% formaldehyde, and incubate at room temperature for 10–30 minutes.
(4) At room temperature, wash the fixed cells with PBS 2–3 times, each time for 10 minutes.
(5) (Optional): At room temperature, permeabilize the sample for 3–5 minutes with a 0.1% Triton X-100 solution dissolved in PBS to enhance its permeability.
(6) At room temperature, wash the cells with PBS 2–3 times, 10 minutes each time.
(7) Add 100 μL of Alexa Fluor 488‑labeled phalloidin working solution per well (96-well plate), and incubate at room temperature in the dark for 20–90 minutes; the incubation time may be shorter in summer.
(8) Wash the cells three times with PBS, 5 minutes each time, to remove excess Alexa Fluor 488–labeled phalloidin.
(9) (Optional): Stain the cell nuclei with an adequate volume of ready-to-use DAPI solution, e.g., 100 μL per well (96-well plate), for 3–5 minutes at room temperature. Wash the cells twice with PBS, 5 minutes each time.
(10) Perform fluorescence imaging using a fluorescence microscope or a confocal microscope, selecting either the Alexa Fluor™ 488 excitation/emission filter set and/or the DAPI excitation/emission filter set (Ex/Em = 364/454 nm).
Alexa Fluor 488–labeled phalloidin can be used to label and detect neuronal surface receptors. It can also be employed to label and detect cell-surface immune molecules, thereby facilitating studies of the activation and regulatory mechanisms of immune cells.
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