一.

主要來(lái)自這篇https://microbeonline.com/acridine-orange-staining-principle-procedure-results-applications/

1. 簡(jiǎn)介

膜透性的吖啶橙插入/結(jié)合到核酸（DNA/RNA），發(fā)出不同的光。和dsDNA結(jié)合發(fā)綠光（520nm），和ssDNA/RNA結(jié)合，發(fā)出紅色熒光（650nm）。這種結(jié)合主要是通過(guò)吖啶分子和核酸堿基的靜電相互作用。吖啶橙是陽(yáng)離子染料，所以也可以和細(xì)胞內(nèi)的低pH部分如溶酶體結(jié)合，發(fā)出橘色光（orange light）。具有熒光異色性。

2.步驟

熒光顯微鏡觀察

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.

流式檢測(cè)

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大學(xué)的植物細(xì)胞生理學(xué)家 Siegfried Strugger 發(fā)現(xiàn)吖啶橙??????有將活細(xì)胞和死細(xì)胞染成不同顏色的獨(dú)特性能??
• Strugger 發(fā)現(xiàn)AO在不同pH條件下可以 辨別植物細(xì)胞的死活：pH為5.7-8.0時(shí)，??活細(xì)胞顯綠色??而死細(xì)胞顯紅色；在??pH大于4.7時(shí) ??,?? AO能 使 死 細(xì) 胞 的 細(xì) 胞 質(zhì) 顯 紅 色 ?? 在 低 ??pH時(shí) ??細(xì) 胞 核 顯 紅 色 ；當(dāng) ??pH 升 到6.8時(shí) ??，開(kāi)始變成黃綠色??。
• AO的熒光異色現(xiàn)象主要是由 于染料單體??二聚體和多聚體等的形成而導(dǎo)致的??,?? AO和核酸可以形成各種復(fù)合物??包括染料分子插入DNA, 堿基之間??發(fā)綠光??以及染料和核酸表面的磷酸根作用??（堆集 ????）
• 由于癌細(xì)胞中核染色質(zhì)及RNA的含量增多??用 ,?? 染色法可以很明顯地看到癌細(xì)胞和正常細(xì)胞的顏色差異
  【參考：細(xì)胞生物學(xué)熒光技術(shù)原理和應(yīng)用chapter3】