Vinculin Stain (Focal Adhesions)

From LPDwiki

About this protocol: This protocol outlines the steps and materials involved in staining samples for vinculin using anti-vinculin antibodies.

Necessary Materials

1. Primary anti-Tubulin-BIII antibody

  • Purchased from Invitrogen: can be purchased from many different hosts.
  • Upon arrival should be aliquoted to 10 uL and stored at -20°C.

2. Secondary antibodies

  • Purchased from Invitrogen: can be purchased from many different hosts against the host of the primary antibody.
  • Upon arrival store at 4°C.

3. 0.1% Triton X-100 Wash Buffer

4. 0.05% Tween20 Wash Buffer

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

6. 5% Goat Serum Blocking Buffer

  • Note: The secondaries I use are from a goat host so this protocol uses goat serum as the blocking serum.

7. DAPI

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

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

Procedure

Primary Antibody Staining Solution Preparation

For 1 mL of 1:100 dilution staining solution

1. Add 990 uL of blocking buffer to a centrifuge tube.

2. Add 10 uL of the primary antibody into the blocking buffer.

3. Mix briefly via pipetting.

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

5. Label with: 1:100 Tubulin-BIII Primary

Secondary Antibody Staining Solution Preparation

For 1 mL of 1:100 dilution staining solution

1. Add 990 uL of PBS[+] to a centrifuge tube.

2. Add 10 uL of the fluorescently conjugated secondary antibody into the PBS[+].

3. Mix briefly via pipetting.

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

5. Label with: 1:100 secondary antibody 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

DIV7 Primary rat neuron-glia culture on glass - Stained with Goat anti-Mouse Alexa Fluor 488 Secondaries and Mouse anti-vinculin Primaries

1. Wash twice with 0.05% Tween20 wash buffer (5 minutes per wash)

2. Wash samples with 0.1% Triton X-100 wash buffer for 3 minutes.

3. Wash twice with 0.05% Tween20 wash buffer (5 minutes per wash).

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 primary antibody staining solution onto each sample (enough to fill only the top of the sample).

9. Incubate for one hour at room temperature.

10. Remove the primary antibody staining solution from each sample.

11. Wash three times with 0.05% Tween20 wash buffer (5 minutes/10 minutes/10 minutes).

12. Transfer samples into a new non-tissue culture treated well.

13. Pipette ~80 uL of secondary antibody staining solution onto each sample (enough to fill only the top of the sample).

14. Incubate with secondary antibody staining solution for one hour.

15. Remove the secondary antibody staining solution from each sample.

16. Wash three times with PBS[+].

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

18. Incubate with DAPI staining solution for 5 minutes.

19. Remove the DAPI staining solution from each sample.

20. Wash three times with 0.05% Tween20 wash buffer.

21. 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.