Vitha S, Baluska F, Braun M, Samaj J, Volkmann D, Barlow P W
Institute of Plant Molecular Biology, Academy of Sciences of Czech Republic, Ceské Budejovice.
Histochem J. 2000 Aug;32(8):457-66. doi: 10.1023/a:1004171431449.
For walled plant cells, the immunolocalization of actin microfilaments, also known as F-actin, has proved to be much trickier than that of microtubules. These difficulties are commonly attributed to the high sensitivity of F-actin to aldehyde fixatives. Therefore, most plant studies have been accomplished using fluorescent phallotoxins in fresh tissues. Nevertheless, concerns regarding the questionable ability of phallotoxins to bind the whole complement of F-actin necessitate further optimization of actin immunofluorescence methods. We have compared two procedures: (1) formaldehyde fixation and (2) rapid freezing and freeze substitution (cryofixation), both followed by embedding in low-melting polyester wax. Actin immunofluorescence in sections of garden cress (Lepidium sativum L.) root gave similar results with both methods. The compatibility of aldehydes with actin immunodetection was further confirmed by the freeze-shattering technique that does not require embedding after aldehyde fixation. It appears that rather than aldehyde fixation, some further steps in the procedures used for actin visualization are critical for preserving F-actin. Wax embedding, combined with formaldehyde fixation, has proved to be also suitable for the detection of a wide range of other antigens.
对于有细胞壁的植物细胞，肌动蛋白微丝（也称为F-肌动蛋白）的免疫定位已被证明比微管的免疫定位要棘手得多。这些困难通常归因于F-肌动蛋白对醛类固定剂的高敏感性。因此，大多数植物研究是在新鲜组织中使用荧光鬼笔环肽完成的。然而，由于担心鬼笔环肽结合F-肌动蛋白全部成分的能力存在问题，有必要进一步优化肌动蛋白免疫荧光方法。我们比较了两种方法：（1）甲醛固定和（2）快速冷冻和冷冻置换（低温固定），两种方法之后均包埋于低熔点聚酯蜡中。用这两种方法对水田芥（Lepidium sativum L.）根切片进行肌动蛋白免疫荧光检测，结果相似。醛类固定后不需要包埋的冷冻破碎技术进一步证实了醛类与肌动蛋白免疫检测的兼容性。看来，对于F-肌动蛋白的保存而言，肌动蛋白可视化所用方法中的一些后续步骤比醛类固定更为关键。事实证明，蜡包埋结合甲醛固定也适用于多种其他抗原的检测。
