Reagents and Equipment:
Same as Protocol 3 above with the following additions/exceptions
- CB buffer (10 mM MES pH 6.1, 138 mM KCl, 3 mM MgCl2, 2 mM EGTA)
- NaBH4 (sodium borohydride)
- Tissue slices rather than tissue culture cells
- Prepare collagen-coated polyacrylamide gels as described above.
- Cross-link 1-3 mg/ml collagen to the polyacrylamide gel for 4 hrs at 4°C using sulfo-SANPAH.
- Polymerize collagen at 37°C in a humidified chamber (37°C, 5% CO2 and 95% humidity) for at least 6 hrs followed by multiple rinsings with warm medium to remove uncross-linked collagen.
- Prepare tissue slice cultures from the region of interest of control and experimental animals.
- Place slices on the collagen matrix, positioning cells of interest toward the collagen if applicable.
- Secure each tissue slice to the collagen matrix with either dialysis tubing or by polymerizing 1.6 mg/ml rat tail collagen I (in medium) on top of the slice.
- Grow and experimentally treat under tissue culture conditions as chosen by experimenter. Migrating cells will move away from the polyacrylamide gel substrate.
Phalloidin Staining Protocol:
- Reconstitute phalloidin according to manufacturer’s directions. Dilute to working concentration as directed. Keep working stock of fluorescent phalloidin in the dark at room temperature.
- Remove collagen matrices containing cells from dishes, cut into smaller pieces, and place into 12 well dishes.
- Pieces of matrices are fixed in 4% paraformaldehyde (CB buffer containing 0.1% Triton X-100) for 30 min at room temperature.
- Rinse fixed tissue with CB buffer + 0.2% Triton X-100 for 30 min at room temperature.
- Optional: Reduce collagen matrix autofluorescence with 2 x 10 min washes in CB buffer + 0.5 mg/ml NaBH4 (sodium borohydride) at room temperature.
- Block fixed and permeabilized gels in CB buffer + 2% BSA, 1% goat serum, 0.2% Triton X-100 overnight at 4°C.
- If applicable, matrix pieces are then incubated with a primary antibody of interest overnight in blocking buffer and then rinsed in CB buffer + 0.2% Triton X-100 multiple times at room temperature.
- After primary antibody incubation (if performed), fluorescent secondary antibody is added for a 4-6 hrs incubation at 4°C.
- In place of primary and secondary antibodies, or in parallel with the secondary antibody, 0.165 mM phalloidin can be used to stain F-actin (4-6 hrs incubation at 4°C). The phalloidin concentration may need to be determined empirically.
- Matrix pieces are rinsed overnight at 4°C in CB buffer.
- Mount matrix piece on glass slide and gently press with a glass coverslip using a drop of anti-fade mounting medium to prevent photo-bleaching.
- If possible, seal each side of coverslip with nail polish.
- Either view immediately or store the slides in the dark at 4°C for up to 24 hrs.
- Visualize the cells in stained samples with an appropriate fluorescence microscope set-up. For example, a confocal fluorescent microscope with a 63X oil-immersion lenses at the appropriate filter excitation and emission settings.
- For analysis of staining in 3D gels, collection of 5-10 representative maximal projection z-stacks from each sample are useful to determine the average brightness of cells within each image.
For detailed information on these reagents and methods, please see:
- Fischer R.S. et al. 2009. Local cortical tension by myosin II guides 3D endothelial cell branching. Curr. Biol. 19, 260–265.
- Lo C.-M. et al. 2000. Cell movement is guided by the rigidity of the substrate. Biophys. J. 79, 144-152.
- Wang Y.-L. and Pelham, R.J. Jr. 1998. Preparation of a flexible porous polyacrylamide substrate for mechanical studies of cultured cells. Methods Enzymol. 298, 489–496.