Corsetti J P, Way B A, Sparks C E, Sparks J D
Department of Pathology and Laboratory Medicine University of Rochester School of Medicine and Dentistry, New York 14642.
Hepatology. 1992 Jun;15(6):1117-24. doi: 10.1002/hep.1840150624.
Hepatocyte autofluorescence represents a major problem in immunofluorescence studies with fluorescein conjugates because of significant spectral overlap. We describe a method for immunostaining hepatocytes with R-phycoerythrin (a fluorochrome with minimal overlap with autofluorescence) with paraformaldehyde fixation and Triton X-100 permeabilization for better antibody penetration. This method produced both perinuclear (presumed Golgi apparatus) and dispersed, reticular staining (presumed endoplasmic reticulum) in rat hepatocytes in culture stained with a monoclonal antibody to rat apolipoprotein B. Treatment with brefeldin A resulted in loss of apolipoprotein B perinuclear staining and increased reticular immunofluorescence consistent with known properties of brefeldin A (inhibition of protein transport within the secretory pathway by dissolution of Golgi bodies). This suggests that apolipoprotein B epitopes are present in both Golgi bodies and endoplasmic reticulum. To demonstrate the utility of the technique for quantitative studies, static cell cytofluorometry of brefeldin A-treated cells was performed, demonstrating increases in specific immunofluorescence of apolipoprotein B corresponding closely to results estimated by monoclonal antibody radioimmunoassays of cellular homogenates. The technique was then used with flow cytometry of single-cell suspensions of control rat hepatocytes derived from immunostained primary cultures to reveal cell-to-cell heterogeneity of apolipoprotein B epitope expression manifested as apolipoprotein B-negative and positive populations. Results for brefeldin A-treated cells revealed even clearer delineation of heterogeneity as indicated by frank bimodality of the populations, along with not only higher mean apolipoprotein B levels but also a significantly higher proportion of apolipoprotein B-positive cells than in the control.
由于存在显著的光谱重叠，肝细胞自发荧光在使用荧光素缀合物的免疫荧光研究中是一个主要问题。我们描述了一种用R-藻红蛋白（一种与自发荧光重叠最小的荧光染料）对肝细胞进行免疫染色的方法，采用多聚甲醛固定和Triton X-100通透处理以促进抗体更好地穿透。用抗大鼠载脂蛋白B单克隆抗体对培养的大鼠肝细胞进行染色后，该方法产生了核周（推测为高尔基体）染色以及分散的网状染色（推测为内质网）。用布雷菲德菌素A处理导致载脂蛋白B核周染色消失，网状免疫荧光增加，这与布雷菲德菌素A的已知特性（通过溶解高尔基体抑制分泌途径中的蛋白质转运）一致。这表明载脂蛋白B表位同时存在于高尔基体和内质网中。为了证明该技术在定量研究中的实用性，对用布雷菲德菌素A处理的细胞进行了静态细胞荧光测定，结果表明载脂蛋白B的特异性免疫荧光增加，与通过细胞匀浆的单克隆抗体放射免疫测定估计的结果密切对应。然后将该技术用于对来自免疫染色原代培养物的对照大鼠肝细胞单细胞悬液进行流式细胞术分析，以揭示载脂蛋白B表位表达的细胞间异质性，表现为载脂蛋白B阴性和阳性群体。对用布雷菲德菌素A处理的细胞的结果显示，群体的明显双峰性表明异质性更加清晰，不仅平均载脂蛋白B水平更高，而且载脂蛋白B阳性细胞的比例也比对照显著更高。
