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AMINO ACID NAPHTHYLAMIDASE METHOD
FOR BILE CANALICULI
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FIXATION: Smears and touch preparations are fixed in ice cold acetone and chloroform 1:1.
TECHNIQUE: Smears of liver aspirates or touch imprints of liver.
SOLUTIONS:
0.1M Acetic Acid
Acetic acid ---------------------------------------------------------------------- 2.8 ml
Distilled water to make a total of ------------------------------------------------ 500.0 ml
0.1M Sodium Acetate
Sodium acetate, anhydrous ----------------------------------------------------- 4.1 gm
Distilled water to make a total of ----------------------------------------------- 500.0 ml
0.1M Acetic acid-Sodium acetate pH 6.5
0.1M acetic acid ----------------------------------------------------------------- 2.0 ml
0.1M sodium acetate ------------------------------------------------------------ 198.0 ml
Check with pH meter and adjust to pH 6.5 with either acetic acid
or sodium acetate solution.
0.85% Sodium Chloride
Sodium chloride ------------------------------------------------------------------ 0.85 gm
Distilled water -------------------------------------------------------------------- 100.00 ml
0.02M Potassium Cyanide
Potassium cyanide --------------------------------------------------------------- 0.65 gm
Distilled water -------------------------------------------------------------------- 100.00 ml
L-alanyl-4-methoxy-2-naphthylamide (AMN) Solution
L-alanine-4-methoxy-2-naphthylamide -------------------------------------------- 0.02 gm
Distilled water -------------------------------------------------------------------- 2.5 ml
0.3% Sodium Borate
(see PAS Method)
1. Place smears in an ice cold solution of equal parts of acetone and chloroform for 3 minutes.
2. Remove and allow the acetone and chloroform to evaporate form the slides.
3. Incubate the smears in the following solution at 37˚ C for one hour:
0.1M acetic acid - sodium acetate buffer --------------- 25.0 ml
0.85% sodium chloride -------------------------------- 20.0 ml
AMN solution ----------------------------------------- 2.5 ml
0.02M potassium cyanide ----------------------------- 2.5 ml
Fast red B salt ----------------------------------------- 25.0 mg
4. Rinse in four changes of distilled water.
5. Counterstain with acidified Lillie-Mayer hematoxylin for 1 minute.
6. Rinse in three changes of distilled water.
7. Blue hematoxylin in 0.3% sodium borate for 15 seconds.
8. Rinse in four changes of distilled water.
9. Mount with glycerine jelly.
Bile canaliculi appear as a regular network of red bands.
Wasastjerma, C. and Ekelund, P.: The amino acid naphthylamidase reaction of the bile canaliculi in liver smears. Acta Cytologica 18:23-29, 1974.
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