Description
Hitsochemical staining of an adherent cell population to determine the covered area of cells that exhibit the alkaline phosphatase (ALP) enzyme and those areas that have mineralized. The ALP positive areas appear red, while the mineralized areas are brown to black in colour. Bone nodules stain highly for ALP and once mature, exhibit mineralization.
Reagents
• PBS (TCMP Fac. Med. U of T)
• 10% NFB:
▪ formalin/formaldehyde 100mL (Fisher Scientific, F79 1)
▪ Na2HPO4 16g (Fisher Scientific, S374 1)
▪ NaH2PO4.H2O 4g (Fisher Scientific, S369 1)
▪ Distilled water 1L
• 2.5% Silver Nitrate solution:
▪ AgNO3 2.5g (Sigma, S8157)
▪ Distilled water 100mL
• Naphthol AS MX-PO4 (Sigma, N5000)
• N,N-Dimethylformamide (DMF) (Fischer Scientific, D1191)
• Tris-HCl MW=157.6 pH 8.3 0.2M
• Red Violet LB salt (Sigma F1625)
• Distilled water
• Sodium Carbonate formaldehyde:
▪ formalin/formaldehyde 25mL (Fisher Scientific, F79 1)
▪ Na2CO3 5g (EM Science, B10240)
▪ Distilled water 100mL
Method
1. Suction off medium from dish, rinse once in cold PBS.
2. Fix in 10% cold Neutral Formalin Buffer (NFB) for 15 min.
3. Suction off all buffer and rinse the dish once with distilled water, then leave in distilled water for 15 min.
4. While waiting, prepare fresh substrate:
a. Naphthol AS MX-PO4 0.005 g
b. DMF (N,N-Dimethylformamide) 200 μL
c. Tris-HCl MW = 157.6 0.2 M pH 8.3 25 mL
d. Distilled water 25 mL
e. Red Violet LB salt 0.03 g
In eppendorf tube, dissolve (a) in DMF. Add to graduated cylinder with (c + d). Add salt (e) to solution (a+b+c+d) and filter with Whatman’s No.1 filter paper immediately prior to adding to dishes to be stained.
5. Incubate dishes for 45 minutes at room temperature.
6. Rinse in distilled water 3-4 times and leave in distilled water 1 hour (not absolutely necessary, can go to next step right after last rinse).
7. Stain with 2.5% silver nitrate for 30 min.
8. Suction off all the silver nitrate and rinse with distilled water 3 times.
9. Cover up the stained cells with distilled water for a while. Then rinse once more with distilled water to eliminate all silver nitrate remain and either examine or set to dry.
Alternative ending:
10. Prior to examination or drying dish, the colour of the mineralized nodules can be deepened by adding sodium carbonate formaldehyde to the dish for 30s – 2min. Care must be taken in the reaction time allowed as the colour may become too intense and turn dish very black, making it hard to read.
11. Suction off all the sodium carbonate formaldehyde and rinse with slowly running tap water.
12. Leave the stained cells in tap water for 1 hour and then rinse again and cover in tap water for counting or set to dry.