Cold Spring Harb Protoc. 2020 Aug 3;2020(8):099689. doi: 10.1101/pdb.prot099689.
For cell staining, fixation methods decrease generally into two classes, organic solvents and cross-linking reagents. Organic solvents such as alcohols and acetone remove lipids and dehydrate the cells, precipitating the proteins on the cellular architecture. Cross-linking reagents such as paraformaldehyde form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens. Choosing between fixation in organic solvents or cross-linking agents is empirical. There are no general rules to decide between the two and both procedures are described here. Both methods may denature protein antigens, and for this reason, antibodies prepared against denatured proteins may be more useful for cell staining. In some instances, anti-denatured-protein antibodies are the only ones that can work. Fixation in protein cross-linking reagents such as paraformaldehyde or glutaraldehyde preserves cell structure better than organic solvents but may reduce the antigenicity of some cell components. Simple fixation with paraformaldehyde or glutaraldehyde does not allow the antibody to access the specimen and therefore is followed by a permeabilization step using an organic solvent or nonionic detergent. Using the organic solvent is easy, but it can destroy certain elements of the cell architecture, although prior fixation with paraformaldehyde does help to preserve the cellular structure. If preservation of cell structure is important, the best first choice would be to use a nonionic detergent.
对于细胞染色，固定方法通常分为两类，有机溶剂和交联试剂。有机溶剂如醇类和丙酮去除脂质并使细胞脱水，使细胞结构上的蛋白质沉淀。交联试剂如多聚甲醛形成分子间桥，通常通过游离氨基，从而形成连接抗原的网络。在有机溶剂或交联剂之间进行选择是经验性的。没有一般规则可以决定两者之间的选择，这里都有描述。这两种方法都可能使蛋白质抗原变性，因此，针对变性蛋白制备的抗体可能更适合用于细胞染色。在某些情况下，只有抗变性蛋白抗体才能起作用。用多聚甲醛或戊二醛等蛋白质交联试剂固定比有机溶剂更好地保存细胞结构，但可能会降低某些细胞成分的抗原性。简单地用多聚甲醛或戊二醛固定并不能使抗体接触到标本，因此需要使用有机溶剂或非离子去污剂进行渗透化处理。使用有机溶剂很简单，但它可能会破坏细胞结构的某些元素，尽管先用多聚甲醛固定有助于保存细胞结构。如果细胞结构的保存很重要，那么最好的首选是使用非离子去污剂。
