FIXATION: 10% buffered neutral formalin.
TECHNIQUE: Paraffin sections cut at 5 µm.
SOLUTIONS:
1% Acetic Acid
Acetic acid ----------------------------------------------------------------------- 1.0 gm
Distilled water -------------------------------------------------------------------- 99.0 ml
0.067M Phosphate Buffer, pH 6.0
Potassium phosphate, monobasic ---------------------------------------------- 3.87 gm
Sodium phosphate, dibasic ----------------------------------------------------- 0.70 gm
Distilled water to make a total of ----------------------------------------------- 500.00 ml
If necessary, adjust the pH to 6.0 with 1N phosphoric acid or 1N
sodium hydroxide.
0.02% Acridine Orange in Phosphate Buffer
Acridine orange, C.I. 46005 ---------------------------------------------------- 0.02 gm
Phosphate buffer, pH 6.0 ------------------------------------------------------- 100.00 ml
0.1M Calcium Chloride
Calcium chloride, anhydrous --------------------------------------------------- 1.11 gm
Distilled water to make a total of ----------------------------------------------- 100.00 ml
1. Deparaffinize and hydrate to distilled water.
2. Place in 1% acetic acid for 1 minute.
3. Place in distilled water for 1 minute.
4. Stain in 0.02% acridine orange in phosphate buffer for 30 seconds.
5. Place in phosphate buffer, pH 6.0 for 1 minute.
6. Differentiate in 0.1M calcium chloride for 45 seconds.
7. Place in phosphate buffer, pH 6.0 for 1 minute.
8. Mount with phosphate buffer, pH 6.0.
Perl, D.P., and Little, B.N.: Acridine orange - nucleic acid fluorescence, its use in routine diagnostic muscle biopsies. Arch. Neurol. 37:641-644, 1980.
Sarnat, H.B., et al.: A fluorochromic stain for nuclei acids to demonstrate submucosal and myenteric neurons in Hirschsprungs disease. Amer. J. Clin. Path. 83:722-725, 1985.
Image Examples:
Diagnostic Application: