Department of Biology, University of Bergen, Bergen High-Technology Center, NO-5020 Bergen, Norway.
J Immunol Methods. 2010 Oct 31;362(1-2):10-21. doi: 10.1016/j.jim.2010.07.008. Epub 2010 Jul 30.
Different salmon leucocyte subpopulations were identified by immunostaining using rabbit antiserum raised against the salmonid cell line TO derived from head kidney leucocytes in combination with other available immunoglobulins. The rabbit anti-TO cell line serum immunostained all isolated leucocytes from head kidney, peripheral blood and spleen, as shown by analyses of these leucocytes by flow cytometry and by fluorescence microscopy. In cytospin preparations, the staining of salmon leucocytes using rabbit anti-TO serum as the primary antibody revealed greater morphological details compared to conventional staining procedures, especially among isolated spleen leucocytes where cells with a morphology usually limited to dendritic cells were seen. Other cells of various shapes and protrusions were also stained although the anti-TO serum did not stain protrusions on all cell types. Among the immunoglobulin positive cells, the thin protrusions were only seen when immunostained using anti-IgM antibody. The same was observed for neutrophils stained using the monoclonal E3D9 antibody. The double staining of cells using rabbit anti-TO serum and monoclonal antibodies specific for IgM positive cells or neutrophils clearly show how the morphology of these cells can be compared with the rest of the leucocyte population. The staining of salmon leucocytes by antiserum to a salmon leucocyte cell line TO provides a tool for staining the total population of salmon immune cells, and can be used in immunofluorescence or confocal microscopy in combinations with labelling of cellular components or pathogens. The detailed morphological characteristics, such as cell protrusions, visualized by the presented staining have not been observed on fish leucocytes by conventional cell staining procedures.
采用免疫染色技术，通过使用兔抗三文鱼头肾白细胞系 TO 的抗血清，结合其他可利用的免疫球蛋白，鉴定出不同的三文鱼白细胞亚群。兔抗 TO 细胞系抗血清可对从头肾、外周血和脾脏中分离出来的所有白细胞进行免疫染色，这可通过流式细胞术和荧光显微镜分析这些白细胞得以证实。在细胞离心涂片上，与传统的染色方法相比，使用兔抗 TO 血清作为一抗对三文鱼白细胞进行染色，可以揭示出更多的形态学细节，尤其是在分离的脾脏白细胞中，可以看到通常局限于树突状细胞形态的细胞。虽然兔抗 TO 血清不能对所有细胞类型的突起进行染色，但也可以对各种形状和突起的其他细胞进行染色。在免疫球蛋白阳性细胞中，只有在用抗 IgM 抗体进行免疫染色时才能观察到薄的突起。使用单克隆 E3D9 抗体对中性粒细胞进行染色时也观察到了同样的情况。使用兔抗 TO 血清和针对 IgM 阳性细胞或中性粒细胞的单克隆抗体对细胞进行双重染色，清楚地显示了如何将这些细胞的形态与白细胞群体的其余部分进行比较。针对三文鱼白细胞系 TO 的抗血清对三文鱼免疫细胞的总群体进行染色，提供了一种工具，可与细胞成分或病原体的标记物结合，用于免疫荧光或共聚焦显微镜。通过本研究提出的染色方法所观察到的三文鱼白细胞的详细形态特征，如细胞突起，在传统的细胞染色方法中尚未观察到。
