STAINING HYDROGELS – PROTOCOL

1. Rinse hydrogels with PBS 1x

2. Fix in 4% PFA (prepared with PBS 1x) 30-60 minutes at RT.

3. Wash with 20 mM glycine prepared with PBS 1x (3x – 5 minutes each).

NOTE: The gels can be stored in PBS + ABM at 4°C (500 µL ABM per 50 mL PBS)

(ABM aliquots in -20°C). If the target protein is intracellular, permeabilize the cells.

4. Incubate with 1% BSA + 20 mM glycine (both prepared with PBS 1x) (1 hour) to block

unspecific binding of the antibodies.

5. Wash the samples with 20 mM glycine prepared with PBS 1x (3x – 5 minutes each).

6. Incubate the samples for 15 minutes in 0.1 % Triton X-100 prepared with PBS 1x.

7. Wash cells with PBS 1x (3x – 5 minutes each).

8. Incubate with the primary antibody diluted in 1% BSA (prepared with PBS 1x)

overnight at 4°C (vinculin, 1:100 rabbit polyclonal).

9. Decant the solution and wash with PBS 1x (4x: 2 minutes, 5 minutes, 15 minutes, 30

minutes).

10. Add the fluorescent dye-labelled secondary antibody (goat anti-rabbit, 1:400) along

with a compatible counterstain for the cytoskeleton (phalloidin alexa 488, 1:1000)

diluted in 1 % BSA and incubate (1 hour at RT protected from light).

11. Decant the secondary antibody solution and wash with PBS 1x (4x: 2 minutes, 5

minutes, 15 minutes, 30 minutes).

12. Incubate with DRAQ5 (1:1000) diluted in PBS 1x (15 minutes at RT protected from

light).

13. Rinse with PBS 1x.