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HomeImaging Analysis & Live Cell ImagingToluidine Blue Staining Protocol for Millicell® Culture Inserts

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.
Materials
Product No.Product NameDescriptionPricing
PIHA01250Millicell® Standing Cell Culture Insertspore size 0.45 μm, diam. 12 mm, white mixed cellulose esters (MCE) membrane, size 24 wells, sterile
PIHA03050Millicell® Standing Cell Culture Insertspore size 0.45 μm, diam. 30 mm, white mixed cellulose esters (MCE) membrane, size 6 wells, sterile
PIHA03050Millicell® Standing Cell Culture Insertspore size 0.45 μm, diam. 30 mm, white mixed cellulose esters (MCE) membrane, size 6 wells, sterile
PICM01250Millicell® Standing Cell Culture Insertspore size 0.4 μm, diam. 12 mm, transparent PTFE membrane, hydrophilic, size 24 wells, sterile
PICM0RG50Millicell® Standing Cell Culture Insertspore size 0.4 μm, diam. 30 mm, transparent PTFE membrane, hydrophilic, H 5 mm, size 6 wells, sterile
PIXP01250Millicell® Standing Cell Culture Insertspore size 12.0 μm, diam. 12 mm, translucent polycarbonate membrane, Tissue Culture (TC)-treated surface, size 24 wells, sterile
PIXP01250Millicell® Standing Cell Culture Insertspore size 12.0 μm, diam. 12 mm, translucent polycarbonate membrane, Tissue Culture (TC)-treated surface, size 24 wells, sterile
PIHP01250Millicell® Standing Cell Culture Insertspore size 0.4 μm, diam. 12 mm, translucent polycarbonate membrane, Tissue Culture (TC)-treated surface, size 24 wells, sterile
PIHP03050Millicell® Standing Cell Culture Insertspore size 0.4 μm, diam. 30 mm, translucent polycarbonate membrane, size 6 wells, Tissue Culture (TC)-treated surface, sterile
PITP01250Millicell® Standing Cell Culture Insertspore size 3.0 μm, diam. 12 mm, translucent polycarbonate membrane, Tissue Culture (TC)-treated surface, size 24 wells, sterile
PI8P01250Millicell® Standing Cell Culture Insertspore size 8.0 μm, diam. 12 mm, translucent polycarbonate membrane, Tissue Culture (TC)-treated surface, size 24 wells, sterile
PSHT004S5Millicell® 96 well Platepore size 0.4 μm, polycarbonate membrane, sterile
PSHT010R5Millicell® 24 well Platepore size 0.4 μm, polycarbonate membrane, sterile
MACAC0RS596-well Collection Plate96-well receiver trays with lids for Millicell®-96 Cell Culture Insert Plates, sterile, pack of 5
PSMW010R524 well Collection Tray24-well receiver trays with lids for Millicell®-24 Cell Culture Insert Plates, sterile, pack of 5
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