Endothelial and macrophage markers employed to distinguish LSEC from KC by immunofluoresence in mouse liver sections.

aThe abbreviations used are: RIIb, Fcγ receptor IIb; MR, mannose receptor; vWF, von Willebrand factor; Cav1, Caveolin 1.bLSEC were defined morphologically in DIC images as a thin cellular layer lining the sinusoidal lumens. KC were definable by the markers employed and by their situation in sinusoids on LSEC. The veins (V), either portal or central, were identifiable by their large luminal size in DIC images. Immunofluorescence intensity was graded on a subjective +/− scale as follows: +++, intense; ++, moderate; +/−, weak and inconsistent; -, no fluorescence above background. Sections of spleen and yolk sac were used as positive controls for antibodies directed at macrophages and endothelial cells, respectively. Note that some endothelial cell markers label the liver veins.cSections of RIIb KO liver show no LSEC fluorescence with mab 2.4G2 anti- FcγRII/III/IV, indicating that only RIIb and not FcγRIII/IV are expressed on LSEC in WT liver; however, KC staining remains +/− in the RIIb-KO liver indicating KC expression of FcγRIII/IV. RIIb expression in KC cannot readily be assessed by this strategy.dThe labeling was not consistent among the triplicate livers with this antibody.eNo consistent labeling within all LSEC and all lobes of liver with this antibody.a. 本文所用缩写如下：RIIb即Fcγ受体IIb（Fcγ receptor IIb）；MR即甘露糖受体（mannose receptor）；vWF即血管性血友病因子（von Willebrand factor）；Cav1即窖蛋白1（Caveolin 1）。 b. 肝窦内皮细胞（liver sinusoidal endothelial cells, LSEC）通过形态学特征在微分干涉相差显微镜（differential interference contrast, DIC）图像中被定义为衬贴于窦状隙管腔的薄层细胞。库普弗细胞（Kupffer cells, KC）可通过所使用的细胞标志物及其在肝窦内皮细胞上方的窦状隙内定位进行识别。门静脉或中央静脉（V）可凭借其在DIC图像中较大的管腔尺寸进行鉴别。免疫荧光强度采用主观±分级标准，具体为：+++代表强阳性；++代表中等强度；+/−代表弱阳性且结果不一致；−代表荧光强度未高于背景。分别以脾脏组织切片和卵黄囊组织切片作为巨噬细胞靶向抗体与内皮细胞靶向抗体的阳性对照。需注意，部分内皮细胞标志物可标记肝脏静脉。 c. RIIb敲除（knockout, KO）小鼠的肝脏组织经抗FcγRII/III/IV单克隆抗体（monoclonal antibody, mab）2.4G2染色后，未检测到肝窦内皮细胞的荧光信号，表明野生型（wild type, WT）小鼠肝脏的肝窦内皮细胞仅表达RIIb，而非FcγRIII/IV；但在RIIb敲除小鼠肝脏中，库普弗细胞的染色仍呈+/−，提示库普弗细胞表达FcγRIII/IV。通过该实验策略无法直接评估库普弗细胞的RIIb表达水平。 d. 采用该抗体进行染色时，三份重复的肝脏组织样本间的标记结果并不一致。 e. 采用该抗体进行染色时，无法在所有肝窦内皮细胞及所有肝叶中获得稳定的标记结果。