Detecting Alkaline Phosphatase–Labeled Cells
Abstract
Different substrates are available for detection of cells labeled with alkaline phosphatase. Naphthol-AS-BI-phosphate (NABP)/New Fuchsin produces an intense red product that is insoluble in alcohols as well as aqueous solutions and is compatible with a range of histochemical stains. Bromochloroindoyl phosphate/nitro blue tetrazolium (BCIP/NBT) is the most commonly used of the chromogenic substrates for alkaline phosphatase. It is somewhat more sensitive than NABP/New Fuchsin but gives less contrast during microscopic observation.
MATERIALS
Reagents
Cells labeled with alkaline phosphatase–labeled antibodies
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Use eukaryotic alkaline phosphatase, which is readily inactivated by EDTA. The bacterial enzyme is difficult to stop, causing overdevelopment and leading to high background.
Reagents for detection using BCIP-NBT (Steps 11–16)
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Bromochloroindolyl phosphate (disodium salt) (BCIP; 0.5 g in 10 mL of 100% dimethylformamide)
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BCIP stock is stable for at least 1 yr at 4°C.
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Nitro blue tetrazolium (0.5 g in 10 mL of 70% dimethylformamide)
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NBT stock is stable for at least 1 yr at 4°C.
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PBS containing 20 mm EDTA
Reagents for detection using NABP-NF (Steps 1–10)
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Dimethylformamide
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EDTA (20 mm)
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HCl (2 n)
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Naphthol AS-TR phosphate (sodium salt)
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New Fuchsin
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Sodium nitrate
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Tris (0.2 m, pH 9.0)
Equipment
Dish or tray to hold cells for staining (for detection with BCIP-NBT)
Shaker (for detection with BCIP-NBT)
METHOD
Detecting Alkaline Phosphatase–Labeled Cells Using NAPB-NF
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1. Dissolve 1 mg of New Fuchsin in 0.25 mL of 2 n HCl.
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2. Dissolve 1 mg of sodium nitrate in 0.25 mL of H2O.
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3. Dissolve 10 mg of naphthol AS-TR phosphate in 0.2 mL of dimethylformamide.
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4. Mix the New Fuchsin solution with the sodium nitrate solution. Shake for 1 min.
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5. Add the mixture to 40 mL of 0.2 m Tris (pH 9.0).
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6. Add the naphthol AS-TR solution to the above mixture.
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7. Apply to specimen and incubate for 10–40 min at room temperature.
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8. Stop the reaction by washing with 20 mm EDTA.
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9. (Optional) Counterstain if necessary (see Protocol: Counterstaining, Mounting, and Photographing Stained Cells [Rodig 2019]).
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10. Mount in DPX (see Protocol: Counterstaining, Mounting, and Photographing Stained Cells [Rodig 2019]).
Detecting Alkaline Phosphatase–Labeled Cells Using BCIP-NBT
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11. Just before developing, prepare fresh substrate solution. Add 66 µL of NBT stock to 10 mL of alkaline phosphatase buffer. Mix well and add 33 µL of BCIP stock. Use within 1 h.
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12. Place the washed cells in a suitable container. Add enough substrate solution to cover the cells (for a 100-mm dish, 3–5 mL is appropriate).
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13. Develop at room temperature with agitation until the stain is suitably dark. Periodic monitoring under the microscope may be necessary for some antigens. A typical incubation would be ∼30 min.
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14. To stop the reaction, rinse with PBS containing 20 mm EDTA, chelating the Mg2+ ions.
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15. (Optional) Counterstain if necessary (see Protocol: Counterstaining, Mounting, and Photographing Stained Cells [Rodig 2019]).
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16. Wash in H2O and mount in DPX (see Protocol: Counterstaining, Mounting, and Photographing Stained Cells [Rodig 2019]).
Footnotes
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From the Antibodies collection, edited by Edward A. Greenfield.
