[Histonet] RE: alkaline phosphatase stain on MMA sections

From:"Driessen, L."

Hello Patty,

I get pretty good result with this protocol.

Alkaline phosphatase:  BCIP-NBT method.
(Manual of Histological Techniques; Bancroft/Cook)

Controlmaterial: kidney, liver, spleen.
Material: Frozen sections, paraffinsections, MMA-sections of (EDTA-decalcified) bone.

- Remove the MMA and bring the slices through alcohol to water.
- Pre-incubate EDTA-decalcified material one night with TRIS/MgCl2-buffer.
- Incubate in alkaline phosphatase medium at 37C (minimal time 30 minutes). Check microscopical.
- Rinse with  water.
- Counterstain with Mayer haematoxylin, 1 minute.
- Rinse with tapwater, 2 minutes.	
- Mount in watermounting medium (NBT disslove in in alcohol, xylene).

Results:
Alkaline phosphatase reactivity: 	Darkblue to black.
	Nuclei:				Blue


TRIS/ MgCl2-buffer:
- 2,4 gram TRIS (hydroxymethylaminomethane).
- 1 gram MgCl2.
- 100 ml distilled water.
Dissolve and bring pH to 9,0 with HCl.

Veronal-buffer:
- 1,9 gram Na-acetate (waterfree).
- 2,9 gram Na-barbiturate (5,5 diethylbarbituurzuur, Na-salt)
- 100 ml distilled water.
Bring pH to 9,5 with 0,1-1N HCl.

1M MgCl2 :
- 20 gram MgCl2 .6H2O.
- 100 ml. distilled water.

Alkaline phosphatase medium: (pH 9,5):
- 12,5 milligram 5-Bromo-4-chloro-3-indolyl phosphate in 1 ml dimethylformamid.
- 50 milliliter 0,2M Veronal-acetattebuffer, pH 9,5
- 25 milligram NBT (Nitrobluetetrazolium).
- 0,4 milliliter 1M Magnesiumchlorid


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