Humenick Adam, Johnson M E, Chen B N, Wee M, Wattchow D A, Costa M, Dinning P G, Brookes S J H
Human Physiology, College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5042, Australia.
Department of Surgery, Flinders Medical Centre, Bedford Park, SA 5042, Australia.
Heliyon. 2024 Feb 21;10(5):e26522. doi: 10.1016/j.heliyon.2024.e26522. eCollection 2024 Mar 15.
Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated secondary antisera) and has been used for sequential staining of sections. The aim of this study was to test whether 2-ME/SDS is effective for eluting indirect immunofluorescent staining (with primary antisera visualised by fluorophore-coupled secondary antisera) in wholemount preparations. We also analysed how 2-ME/SDS may work and used this understanding to devise additional uses for immunofluorescence in the nervous system. 2-ME/SDS appears to denature unfixed proteins (including antisera used as reagents) but has much less effect on antigenicity of formaldehyde-fixed epitopes. Moieties linked by strong biotin-streptavidin bonds are highly resistant to elution by 2-ME/SDS. Two primary antisera raised in the same species can be applied without spurious cross-reactivity, if a specific order of labelling is followed. The first primary antiserum is followed by a biotinylated secondary, then a tertiary of fluorophore-conjugated streptavidin. The preparation is then exposed to 2-ME/SDS, which has minimal impact on labelling by the first primary/secondary/tertiary combination. However, when this is followed by a second primary antiserum (raised in the same species), followed by a fluorophore-conjugated secondary antiserum, the intervening 2-ME/SDS exposure prevents cross-reactivity between primary and secondary antisera of the two layers. A third property of 2-ME/SDS is that it reduces lipofuscin autofluorescence, although it also raises background fluorescence and strongly enhances autofluorescence of erythrocytes. In summary, 2-ME/SDS is easy to use, cost-effective and does not require modified primary antisera. It can be used as the basis of a multi-layer immunohistochemistry protocol and allows 2 primary antisera raised in the same species to be used together.
间接免疫荧光法通常每个样本只能检测3 - 5种标志物，限制了共存分析。含有2 - 巯基乙醇和十二烷基硫酸钠（2 - ME/SDS）的溶液可洗脱间接免疫荧光标记（即先用一抗，再用荧光团偶联的二抗），已用于切片的连续染色。本研究的目的是测试2 - ME/SDS在整装标本中洗脱间接免疫荧光染色（用荧光团偶联的二抗检测一抗）是否有效。我们还分析了2 - ME/SDS的作用机制，并利用这一认识设计了在神经系统中免疫荧光的其他用途。2 - ME/SDS似乎能使未固定的蛋白质（包括用作试剂的抗血清）变性，但对甲醛固定表位的抗原性影响较小。通过强生物素 - 链霉亲和素键连接的部分对2 - ME/SDS洗脱具有高度抗性。如果按照特定的标记顺序，在同一物种中产生的两种一抗可以同时使用而不会出现假交叉反应。先用第一种一抗，然后是生物素化二抗，再是荧光团偶联的链霉亲和素三抗。然后将标本暴露于2 - ME/SDS中，其对第一种一抗/二抗/三抗组合的标记影响最小。然而，在此之后加入第二种一抗（同一物种产生），再加入荧光团偶联的二抗时，中间的2 - ME/SDS处理可防止两层一抗和二抗之间的交叉反应。2 - ME/SDS的第三个特性是它能降低脂褐素自发荧光，尽管它也会增加背景荧光并强烈增强红细胞的自发荧光。总之，2 - ME/SDS易于使用、成本效益高，且不需要修饰一抗。它可作为多层免疫组织化学方案 的基础，并允许在同一物种中产生的两种一抗同时使用。
