Phalloidin CruzFluor™ 594 Conjugate is a red fluorescent conjugate that is an excellent substitute for AlexFluor® 594. Phalloidin is a heptapeptide phallotoxin produced by Amanita phalloides, the death cap mushroom. Phalloidin binds and stabilizes filamentous F-actin, preventing the dissociation of paired filaments and disturbing the natural equilibrium between filamentous (polymeric) and globular (monomeric) actin in the cell, as well as inhibit the ATP hydrolysis activity of F-actin. Actin is used by the cell for mechanical processes such as growth, scaffolding of the cytoskeleton, and locomotion, and disruption of this actin equilibrium destroys cellular functioning. The magnitude by which the actin protein differs from species to species is no more than 20%. These properties of Phalloidin make Phalloidin CruzFluor™ Conjugates a fantastic tool for labeling, identifying and quantitating F-actins. The Phalloidin CruzFluor™ Conjugates are used at nanomolar concentrations with cell-free experiments, cell culture experiments, formaldehyde-fixed, permeabilized tissue sections and the study of actin networks. Because Phalloidin CruzFluor™ Conjugates are offered in different colors the series is excellent for multicolor imaging applications.
Phalloidin CruzFluor™ conjugates can be used alongside antibodies in IF staining of formalin-fixed cells and formalin-fixed, frozen tissue sections. The Phalloidin will stain F-actin.
1. Prepare 1000 X Phalloidin DMSO stock solution: by adding 30 uL of DMSO into the powder form vials.
2. Prepare 1X Phalloidin conjugate working solution: by adding 1 uL of 1000X Phalloidin conjugate DMSO solution to 1 mL of PBS with 1% BSA
Note 1: The unused 1000X DMSO stock solution of Phalloidin conjugate should be aliquoted and stored at -20° C. protected from light.
Note 2: Different cell types might be stained differently. The concentration of Phalloidin conjugate working solution should be prepared accordingly.
3. Stain the cells or tissue:
3.1 Perform formaldehyde fixation. Incubate cells with 3.0-4.0% formaldehyde in PBS at room temperature for 10-30 minutes.
Note 3: Avoid any methanol containing fixatives since methanol can disrupt actin during the fixation process. The preferred fixative is methanol-free formaldehyde.
3.2 Rinse the fixed cells 2-3 times in PBS.
3.3 Optional: Add 0.1% Triton X-100 in PBS into fixed cells (from Step 3.2) for 3 to 5 minutes to increase permeability. Rinse the cells 2-3 times in PBS.
3.4 Add Phalloidin conjugate working solution (from Step 2) into the fixed cells (from Step 3.2 or 3.3), and stain the cells at room temperature for 20 to 90 minutes.
3.5 Rinse cells gently with PBS 2-3 times to remove excess Phalloidin conjugate before mounting, sealing and imaging under microscope.
Note 4: If antibody staining is desired, perform blocking, primary antibody and secondary antibody incubations separately between Steps 3.3 and 3.4 according to manufacturer's protocol. Labeling with Phalloidins can be combined with secondary antibody staining, as long as the correct dilutions of each reagent are used in the staining solution.
Note 5: Paraffin-embedded tissues are not recommended for Phalloidin staining, because the use of solvents during the deparaffinization process will disrupt actin.
1000X stock solution in DMSO; 30 µl total
Soluble in DMSO.
Store at -20° C
λex 590 nm, λem 618 nm
For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.
UN 2811, Class 6.1, Packing group II
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Rated 5 out of
Very good resultIt worked perfectly in AGS cells. The cells were incubated with Phalloidin CruzFluor 594 in 1% BSA in PBST (1:1000) for 1 hour at RT. DAPI was used to counterstain the cell nuclei. Digital images of cells were acquired with a Zeiss Axio Vert.A1 fluoresecence microscope.
Date published: 2018-01-29
Rated 5 out of
work wellThis product worked well in concentration of 1:800 and incubating for 1h at 37℃ in darkness. The cells were osteoclasts from mouse bone.
Date published: 2017-05-31
Rated 5 out of
Strong red fluorescence staining was observedStrong red fluorescence staining was observed by IF following incubation of formalin fixed HeLa cells with a F-actin antibody conjugated to Phalloidin CruzFluor 594 Conjugate. Crisp red fluorescence observed. -SCBT QC
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