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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.
