Toluidine Blue Staining Protocol for Millicell®

Materials

  1. Millicell® inserts or insert plates
  2. Milli-Q® water
  3. Millex®-GP filter unit — Millipore cat. no. SLGP033RS
  4. Toluidine blue (Sigma)
  5. 3% glutaraldehyde in Phosphate Buffered Saline (PBS)
  6. Triton X-100 (Sigma), 0.5% Method

Method

  1. Prepare a 0.3 % solution (gram percent) of toluidine blue in Milli-Q® water, stir, and filter through a Millex-GP filter unit.
  2. Remove the Millicell® insert from the plate and wash gently with PBS to remove growth media.
  3. Fix the cells for 15 minutes with 3% glutaraldehyde in PBS.
  4. Rinse gently with Milli-Q® water. Repeat twice
  5. Permeabilize the cells with 0.5% Triton X-100 for 5 minutes.
  6. Rinse gently with Milli-Q® water. Repeat twice.
  7. Apply stain to the apical cell side of membrane for 30–60 seconds.
  8. Observe as a wet mount.

Materials