Hardham A R
J Histochem Cytochem. 1985 Feb;33(2):110-8. doi: 10.1177/33.2.3918095.
The study of the surface properties of zoospores and cysts of the fungus Phytophthora cinnamomi required a fixation regime that would preserve the cells adequately and not interfere with binding and detection of probes on the cell surface. When they were fixed in 4% formaldehyde (F), specific binding of concanavalin A-fluorescein isothiocyanate and rhodamine-labeled soybean agglutinin was obtained. However, electron microscopy showed that preservation was so poor that intracellular binding sites had become exposed. By contrast glutaraldehyde (G), even at concentrations as low as 0.05%, gave good preservation of the zoospores but induced high levels of nonspecific fluorescence, making its use impractical for studies using fluorescent probes. Addition of 1-4% F to 0.05-0.8% G reduced the level of G-induced fluorescence while not diminishing the quality of ultrastructural preservation. This effect was evaluated quantitatively and an optimum fixation regime for the fungal cells, namely, 0.2% G and 2-4% F in 50 mM PIPES buffer, was determined. This combined fixative facilities correlated fluorescence and ultrastructural labeling with lectins and immunocytochemical probes.
对樟疫霉菌游动孢子和孢囊表面特性的研究需要一种固定方法，该方法既能充分保存细胞，又不会干扰细胞表面探针的结合与检测。当它们用4%甲醛（F）固定时，可获得伴刀豆球蛋白A-异硫氰酸荧光素和罗丹明标记的大豆凝集素的特异性结合。然而，电子显微镜显示保存效果很差，细胞内结合位点已暴露。相比之下，戊二醛（G）即使浓度低至0.05%，也能很好地保存游动孢子，但会产生高水平的非特异性荧光，使其不适用于使用荧光探针的研究。在0.05 - 0.8% G中添加1 - 4% F可降低G诱导的荧光水平，同时不降低超微结构保存的质量。对这种效果进行了定量评估，并确定了真菌细胞的最佳固定方法，即在50 mM PIPES缓冲液中使用含0.2% G和2 - 4% F的固定液。这种联合固定剂有助于将荧光和超微结构标记与凝集素和免疫细胞化学探针相关联。
