190402 吖啶橙（Acridine Orange）染色原理、步骤

一.

主要来自这篇https://microbeonline.com/acridine-orange-staining-principle-procedure-results-applications/

1. 简介

膜透性的吖啶橙插入/结合到核酸（DNA/RNA），发出不同的光。和dsDNA结合发绿光（520nm），和ssDNA/RNA结合，发出红色荧光（650nm）。这种结合主要是通过吖啶分子和核酸碱基的静电相互作用。吖啶橙是阳离子染料，所以也可以和细胞内的低pH部分如溶酶体结合，发出橘色光（orange light）。具有荧光异色性。

2.步骤

荧光显微镜观察

Requirements: Acridine orange,Glacial Acetic acid, Distilled water
Preparation of reagent: 50 mg acridine orange is dissolved in 10 ml of distilled water to preparea stock solution and stored in the refrigerator.1 ml of Acridine orange stock solution and 0.5 ml of glacial acetic acid is added to 50 ml of distilled water to prepare a working solution.
Staining procedure:
Prepare a smear in a clean grease free slide and allow it to air dry.
The slide is then fixed with methanol and dried again.
It is then put in trough with acridine orange staining working solution (i.e 0.01 per cent).
After 2 minutes of staining, the slides are washed gently with water and dried and then examined in a fluorescent microscope.
Observance: Bacteria stain orange against a green to yellow background of human cells and debris.

流式检测

Requirements: 0.1M Citric Acid (dissolve 1.921g per 100ml distilled water) ,0.2M Dibasic Sodium Phosphate (dissolve 2.839g per 100ml distilled water) ,Triton X-100 (Baker),0.5M EDTA, Sodium chloride(NaCl), Acridine Orange (Powder) and Sucrose.
Preparation of reagents:

• Stock Buffer I :20mM Citrate-Phosphate, pH 3.0, 0.1mM EDTA, 0.2M Sucrose, 0.1% Triton X-100
  (To 125ml distilled water add 40µl 0.5M EDTA, 26.48ml 0.1M Citric Acid, 6.85ml 0.2M Dibasic Sodium Phosphate, 13.69g Sucrose, 0.2ml Triton X-100 .QS to 200ml and 0.2µ filter. Store at 4oC)
• Stock Buffer II :10mM Citrate-Phosphate, pH 3.8, 0.1M NaCl
  (To 150 ml distilled water add 9.92ml 0.1M Citric Acid, 5.46ml 0.2M Dibasic Sodium Phosphate, 1.7g NaCl. QS to 200ml and 0.2m filter. Store at 4oC)
  Staining Procedure:
• Make a 2mg/ml solution of Acridine orange in distilled water and dilute to 1:100 in Buffer II
• Aliquot cells: 105- 106 in 100µl PBS or media .
• Add Buffer I (0.5ml) at room temp, agitate to suspend .
• Add Buffer II + AO (0.5ml) at room temp, agitate to suspend.
• Run on flow cytometer. Excitation 488 nm; dot plot of green fluorescence at 530nm versus red fluorescence >600 nm).

二

• Mundter大学的植物细胞生理学家 Siegfried Strugger 发现吖啶橙有将活细胞和死细胞染成不同颜色的独特性能
• Strugger 发现AO在不同pH条件下可以 辨别植物细胞的死活：pH为5.7-8.0时，活细胞显绿色而死细胞显红色；在pH大于4.7时 , AO能 使 死 细 胞 的 细 胞 质 显 红 色 在 低 pH时 细 胞 核 显 红 色 ；当 pH 升 到6.8时 ，开始变成黄绿色。
• AO的荧光异色现象主要是由 于染料单体二聚体和多聚体等的形成而导致的, AO和核酸可以形成各种复合物包括染料分子插入DNA, 碱基之间发绿光以及染料和核酸表面的磷酸根作用（堆集 ）
• 由于癌细胞中核染色质及RNA的含量增多用 , 染色法可以很明显地看到癌细胞和正常细胞的颜色差异
  【参考：细胞生物学荧光技术原理和应用chapter3】