ALKALINE PHOSPHATASE

ALKALINE PHOSPHATASE

SECTIONS

Fresh Frozen (jejunal Biopsy) or cold Formal calcium/Gum Sucrose fixed Cryostat 5-7micron thick.

 

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SOLUTIONS

a. 0.2M Tris HCL buffer pH 8.3

• 0.2M Tris (2.42g/100) 25ml 125ml )
  
• 0.1N HCL (0.85/110ml) 20ml 100ml ) 500ml
  
• Distilled water 55ml 275ml )
  

b. Naphthol ASBI stock

• Naphthol ASBI phosphate 25mg
  
• N:N Dimethyl formamide 10ml
  
• Distilled water 10ml
  
• Adjust to pH 8.0 with 1M sodium carbonate if necessary then add 300 ml distilled water
  
• 0.2M Tris HCL buffer pH 8.3 (solution a.) 180ml
  

c. Incubating solution

• Naphthol ASBI stock (solution b.) 10ml
  
• Fast red TR 10mg
  
• Filter, use immediately
  

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METHOD

 

1. Incubate in solution c. for 5-15 minutes at room temperature.
   
2. Wash, counterstain in haematoxylin, wash.
   
3. Mount in phosphate buffered glycerine jelly
   

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RESULTS

 

• Alkaline phosphatase activity - Red
  
• Nuclei - Blue
  

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COMMENTS

• Naphthol ASBI stock keeps well in refrigerator at 4C
  
• Do not over-incubate.
  

NOTE: No responsibility is assumed by The University of Nottingham or the Queens Medical Centre NHS Trust for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. It is the users responsibilty to ensure that all procedures are carried out according to appropriate Health and Safety requirements.