Toluidine Blue Staining Protocol for Millicell®
Materials
- Millicell® inserts or insert plates
- Milli-Q® water
- Millex®- GP filter unit — Product No. SLGP033RS
- Toluidine blue (Sigma)
- 3 % glutaraldehyde in Phosphate Buffered Saline (PBS)
- Triton X-100 (Sigma), 0.5 % Method
Method
- Prepare a 0.3 % solution (gram percent) of toluidine blue in Milli-Q® water, stir, and filter through a Millex-GP filter unit.
- Remove the Millicell® insert from the plate and wash gently with PBS to remove growth media.
- Fix the cells for 15 minutes with 3 % glutaraldehyde in PBS.
- Rinse gently with Milli-Q® water. Repeat twice
- Permeabilize the cells with 0.5 % Triton X-100 for 5 minutes.
- Rinse gently with Milli-Q® water. Repeat twice.
- Apply stain to the apical cell side of membrane for 30–60 seconds.
- Observe as a wet mount.
Materials
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