Jiao Y, Sun Z, Lee T, Fusco F R, Kimble T D, Meade C A, Cuthbertson S, Reiner A
Department of Anatomy and Neurobiology, College of Medicine, The University of Tennessee-Memphis, The Health Science Center, 38163, USA.
J Neurosci Methods. 1999 Nov 15;93(2):149-62. doi: 10.1016/s0165-0270(99)00142-9.
The masking of antigens by aldehyde-containing fixatives or by paraffin embedding procedures is a problem for immunohistochemical studies. Enzymatic digestion, formic acid treatment, microwave heating and autoclave heating have been used to deal with this problem, with microwave heating-based antigen retrieval having become widely used as the method of choice. Microwave heating, however, has the shortcoming that it is difficult to precisely control the heating temperature and it is difficult to apply this method of heating to free-floating sections without damaging the sections. We describe here a simple, reliable and sensitive antigen retrieval method that uses water-bath heating. By this method, the temperature can be precisely controlled to yield effective antigen retrieval with minimal tissue damage in free-floating or paraffin-embedded slide-mounted sections. We found that the best results were obtained with a 30 min incubation in a 10-50 mM sodium citrate solution (pH 8.5-9.0) preheated to and maintained at 80 degrees C in a water-bath, followed by 30 min incubation in 0.3-3% nonfat dry milk to reduce nonspecfic staining. This method is highly effective for both 40 microm free floating sections, slide-mounted cryostat sections and paraffin-embedded slide-mounted sections, and it works well for tissue from diverse species (human, rat, mouse, pigeon, and zebra finch) and for diverse antigens (e.g. enkephalin, substance P, huntingtin, GluR1, GFAP, and ubiquitin). This method was also found to enhance immunolabeling in glutaraldehyde-fixed tissue that had been prepared for ultrastructural examination, without having a deleterious effect on the ultrastructure.
含醛固定剂或石蜡包埋程序对抗原的掩盖是免疫组织化学研究中的一个问题。酶消化、甲酸处理、微波加热和高压灭菌加热已被用于处理这个问题，其中基于微波加热的抗原修复已成为广泛使用的首选方法。然而，微波加热有缺点，即难以精确控制加热温度，并且难以将这种加热方法应用于漂浮切片而不损坏切片。我们在此描述一种使用水浴加热的简单、可靠且灵敏的抗原修复方法。通过这种方法，可以精确控制温度，在漂浮或石蜡包埋的载玻片切片中以最小的组织损伤实现有效的抗原修复。我们发现，将切片在预热至80摄氏度并保持在该温度的水浴中的10 - 50 mM柠檬酸钠溶液（pH 8.5 - 9.0）中孵育30分钟，然后在0.3 - 3%脱脂奶粉中孵育30分钟以减少非特异性染色，可获得最佳结果。该方法对40微米的漂浮切片、冷冻切片载玻片和石蜡包埋载玻片切片均非常有效，并且对来自不同物种（人、大鼠、小鼠、鸽子和斑胸草雀）的组织以及不同抗原（例如脑啡肽、P物质、亨廷顿蛋白、GluR1、GFAP和泛素）均适用。还发现该方法可增强为超微结构检查而制备的戊二醛固定组织中的免疫标记，而对超微结构没有有害影响。
