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Immunofluorescent analysis of Calreticulin (green) U2OS cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA (Product # 37525) in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were probed with a Calreticulin polyclonal antibody (Product # PA3-900) at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:250 for 30 minutes at room temperature. Actin filaments (red) were stained with DyLight 554 Phalloidin (Product # 21834) at a dilution of 1:300 (1unit/ml final concentration) and nuclei (blue) were stained with Hoechst (Product # 62249) at a concentration of 1ug/mL. Images were taken on a Thermo Scientific ArrayScan VTI at 20X magnification.
|Tested species reactivity||Many|
|Host / Isotype||Not Applicable|
|Tested Applications||Dilution *|
|Immunocytochemistry (ICC)||1 - 5 units/ml|
|Immunofluorescence (IF)||1 - 5 units/ml|
|Immunohistochemistry (IHC)||Assay dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Format: 300 units of lyophilized DyLight 554-Phalloidin (300 units/ml) in methanol. Prepare stock solution by adding 1000 µL of pure methanol to the vial and gently mix.
21834 has been successfully used in immunofluorescence and immunohistochemistry. DyLight 554-Phalloidin has an excitation/emission of 551/572nm and molecular weight of 1313.5 g/mole.
DyLight 554-Phalloidin Stock Solution can be prepared in methanol, DMF, or DMSO and unused Stock Solution should be promptly stored at -20°C to minimize evaporation. Working solutions (per 96-well plate) can be prepared by diluting 20 µL of the Stock Solution in 5.98 ml of PBS and mixing well.
Typical staining procedure adds 50µl of Working Solution (i.e. 1 to 5 unit/ml) to a each well. Incubate cells in the dark for 30 minutes at room temperature (optimal staining times varies from 10 minutes to 3 hours depending on cell type). Aspirate and wash cells three times in PBS after incubation. If desired, probe with specific antibodies or dyes before using DyLight 554-Phalloidin.
Phalloidin is a bicyclic peptide that belongs to a family of toxins isolated from the deadly Amanita phalloides “death cap” mushroom and is commonly used as a counterstain (similar to DAPI or Hoechst) in cell biology and histology imaging applications to selectively label F-actin in fixed cells, permeabilized cells, and cell-free experiments. Labeled phalloidin conjugates have similar affinity for both large and small filaments and bind in a stoichiometric ratio of about one phallotoxin per actin subunit in both muscle and non-muscle cells. Phalloidins reportedly do not bind to monomeric G-actin, unlike some antibodies against actin. The dynamics of the actin polymerization in cells are important for a variety of cellular processes from cell motility to cell shape, from muscular contraction to cytokinesis, and more.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Amanita phalloides; DY554-Phalloidin; F-actin toxin