Toluidine Blue Staining Protocol for Millicell^® Culture Inserts

Toluidine blue is a basic thiazine metachromatic dye that binds to nuclear material of tissues with a high DNA and RNA content and is used for selectively staining acidic tissue components such as carboxylates, sulfates, and phosphate radicals. This effective staining agent is used for identifying the development and accumulation of the cartilage matrix, proteoglycans for isolation and culture of mesenchymal stem cells from mouse compact bone.

Materials

• Millicell^® inserts or insert plates
• Milli-Q^® water
• Millex^®- GP filter unit (Product No. SLGP033RS)
• Toluidine blue (Product No. 89640)
• 3 % glutaraldehyde in phosphate-buffered saline (PBS)
• Triton X-100, 0.5 % 

Method for Toluidine Blue Staining

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.