Phalloidin Stain (F-actin)

From LPDwiki

About this protocol: This protocol outlines the steps and materials involved in staining samples for f-actin using pre-conjugated phalloidin.

Necessary Materials

1. Fluorescently Tagged Phalloidin

  • Purchased from Invitrogen: can be purchased with many different tagged fluorescent molecules.
  • Upon arrival should be aliquoted to 20 uL and stored at -20°C.

2. 0.1% Triton X-100 Wash Buffer

3. Phosphate Buffered Saline Solution with Mg+ and Ca+

4. 10% FBS Blocking Buffer

5. DAPI

  • Purchased from Sigma (D9542) and should be stored in 4°C upon arrival.

6. Non-tissue culture treated plates (typically use 6 well)

Procedure

Phalloidin Staining Solution Preparation

For 1 mL of 1:50 dilution staining solution

1. Add 950 uL of PBS[+] to a centrifuge tube.

2. Add 50 uL of the conjugated phalloidin into the PBS[+].

3. Mix briefly via pipetting.

4. Optional: Spin down the tube for 10 seconds.

5. Label with: 1:50 Phalloidin Solution

DAPI Staining Solution Preparation

DIV12 Primary rat neuron-glia co-culture cell nuclei - Stained with DAPI

For 1 mL of 1:20000 dilution staining solution

1. Add 199 uL of PBS[+] to a centrifuge tube.

2. Add 1 uL of DAPI into the PBS[+].

3. Mix briefly via pipetting.

4. Optional: Spin down the tube for 10 seconds.

5. Add 990 uL of PBS[+] into a second centrifuge tube.

6. Add 10 uL of the diluted DAPI solution into the second centrifuge tube.

7. Mix briefly via pipetting.

8. Optional: Spin down the tube for 10 seconds.

9. Label with: 1:20000 DAPI solution

Staining Protocol

NIH/3T3 Fibroblasts stained with Alexa Fluor 488 Phalloidin
NIH/3T3 Fibroblasts stained with Alexa Fluor 555 Phalloidin

Important: If you are using this protocol in conjunction with an antibody staining procedure use the antibody procedure and add the phalloidin incubation in after the secondary antibody incubation.

1. Wash twice with PBS[+].

2. Wash samples with 0.1% Triton X-100 wash buffer for 5 minutes.

3. Wash twice with PBS[+].

4. Transfer samples into a non-tissue culture treated well.

5. Pipette ~80 uL of blocking buffer solution onto each sample (enough to fill only the top of the sample).

6. Incubate with blocking buffer for 30 minutes at room temperature.

7. Remove the blocking buffer from each sample.

8. Pipette ~80 uL of phalloidin staining solution onto each sample (enough to fill only the top of the sample).

9. Incubate for 30 minutes at room temperature.

  • Note: This step should be done in the dark.

10. Remove the phalloidin staining solution from each sample.

11. Wash three times with PBS[+].

12. Pipette ~80 uL of DAPI staining solution onto each sample (enough to fill only the top of the sample).

13. Incubate with DAPI staining solution for 5 minutes.

14. Remove the DAPI staining solution from each sample.

15. Wash three times with PBS[+].

16. Proceed with the general staining protocol to mount the sample.

Cleaning Up

1. Dispose of the used buffers by rinsing down the drain.

2. Dispose of used conical tubes and pipettes into the biological waste bin.

3. Thoroughly rinse out the used bottle and hang up to dry.