Whiteland J L, Shimeld C, Nicholls S M, Easty D L, Williams N A, Hill T J
Department of Ophthalmology, University of Bristol, UK.
J Immunol Methods. 1997 Dec 15;210(1):103-8. doi: 10.1016/s0022-1759(97)00167-1.
We have successfully developed a method for the immunohistochemical detection of interleukin 2 (IL-2), IL-4, IL-6, IL-10O, IFNgamma and TNFalpha using monoclonal antibodies (MAb), in sections of mouse tissue embedded in paraffin wax. The method involved fixation in periodate-lysine-paraformaldehyde (PLP), rapid dehydration and infiltration under vacuum with paraffin wax at 54 degrees C. Comparative observations demonstrated that the method gives equivalent or better results than formaldehyde fixed, frozen sections. Since reliable controls, both positive and negative, are paramount for interpretation of immunohistochemical staining, such controls were determined. The following tissues were shown to be suitable as positive controls when using paraffin-embedding: spleen for the detection of TNFalpha, small intestine for IL-2, IL-4 and IL-10, and HSV-1 infected eyes for IL-6 and IFNgamma. We conclude that PLP fixation and low temperature paraffin-embedding is a method which provides both preservation of excellent tissue morphology and reliable immunohistochemical identification of cytokines. These attributes will be invaluable in a wide variety of experimental situations.
我们已经成功开发出一种方法，可使用单克隆抗体（MAb）在石蜡包埋的小鼠组织切片中进行白细胞介素2（IL-2）、IL-4、IL-6、IL-10O、干扰素γ（IFNγ）和肿瘤坏死因子α（TNFα）的免疫组织化学检测。该方法包括用高碘酸盐-赖氨酸-多聚甲醛（PLP）固定，快速脱水，并在54摄氏度下真空用石蜡渗透。对比观察表明，该方法比甲醛固定的冷冻切片能给出同等或更好的结果。由于可靠的阳性和阴性对照对于免疫组织化学染色的解释至关重要，因此确定了此类对照。当使用石蜡包埋时，以下组织被证明适合作为阳性对照：用于检测TNFα的脾脏，用于检测IL-2、IL-4和IL-10的小肠，以及用于检测IL-6和IFNγ的单纯疱疹病毒1（HSV-1）感染的眼睛。我们得出结论，PLP固定和低温石蜡包埋是一种既能保存出色的组织形态又能可靠地进行细胞因子免疫组织化学鉴定的方法。这些特性在各种实验情况下都将非常宝贵。
