Buchynska L, Kashuba E, Szekely L
Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine, Kiev 03022, Ukraine.
Exp Oncol. 2008 Dec;30(4):327-9.
Crystal violet treatment of formalin fixed paraffin embedded tissue slides greatly reduces the endogenous autofluorescence, and allows immunofluorescence (IF) staining with FITC or Alexa488 conjugated antibodies. Using cold CCD camera to capture the fluorescence images makes this staining method very sensitive. Here we show that combination of IF with the simultaneous recording of crystal violet induced red and Hoechst 33258 induced blue fluorescence permits the localization of the IF signal over a cytoplasmic: nuclear red:blue stain that visualizes the microscopic anatomy of the underlying tissue. To make the visual interpretation of the IF staining easier for microscopists, who are used to DAB staining over weak hematoxilin-eosin background, we created a simple color conversion procedure that turns the captured three-color fluorescence RGB (red, green, blue) images over a black background into four color CMYK (cyan, magenta, yellow, key color (black)) images.
用结晶紫处理福尔马林固定石蜡包埋组织切片可大大降低内源性自发荧光，并允许使用异硫氰酸荧光素（FITC）或Alexa488偶联抗体进行免疫荧光（IF）染色。使用冷电荷耦合器件（CCD）相机捕获荧光图像使这种染色方法非常灵敏。在此我们表明，将IF与结晶紫诱导的红色荧光和Hoechst 33258诱导的蓝色荧光同时记录相结合，可在细胞质:细胞核红色:蓝色染色上定位IF信号，该染色可显示基础组织的微观解剖结构。为了让习惯在淡苏木精-伊红背景上进行二氨基联苯胺（DAB）染色的显微镜工作者更容易对IF染色进行视觉解读，我们创建了一个简单的颜色转换程序，将在黑色背景上捕获的三色荧光RGB（红、绿、蓝）图像转换为四色CMYK（青、品红、黄、关键色（黑））图像。
