Double-labeling immunofluorescence staining

rainoffallingstar 于 2018-11-17 发布

Double-labeling immunofluorescence staining

Method

  1. ⽤ 10 g L 戊巴⽐妥钠腹腔 注射⿇醉后 , 经左⼼室插管 , ⽣理盐⽔灌洗 , 然后⽤ 40 g L 多聚甲醛全⾝灌注固 定 。 取出⼤⿏⼤脑 , 将 其放⼊ 40 g L 多聚甲醛进⾏后固定 24 h 。经 200 g L 蔗糖( 40 g L 多聚 甲醛配制)脱⽔ ,直⾄组 织沉底 。在恒冷箱冰冻切⽚机切⽚ , ⽚厚 10 μ m。[1]

  2. The frozen sections (10-µM thick; frozen at -80˚C) of brain tissues were defrosted, fixed with acetone at 4˚C for 30 min, and washed with PBS （0 . 01 mol/ L？）for 5 min three times. [3]

  3. Antigen retrieval was performed by boiling in antigen retrieval solution I at 100˚C for 5 min, and washed with PBS for 5 min three times. Sections were blocked with bovine serum albumin (BSA； ⽜⾎清蛋⽩？？) at room temperature for 1 h. [3]

切⽚在加⼊⼀抗前 , 最好预先⽤⼆抗同种动物 ⾎清封闭 30 min , ⽬的是为了结合掉⾮特异抗原 。必要 时 , 也可加⽤ 0 . 3 % H 2 O2-甲醇孵育 10 min 以 清除内源性过氧化物酶的活性 。[1]

  1. When the blocking solution was removed, the sections were incubated with primary antibodies against X-protein (rabbit? anti-rat antibody; 1:1,000【最适浓度？】) and X2-protein (mouse？ anti-rat antibody; 1:1,000【最适浓度？】) at 4˚Covernight.[3]

免疫 荧光双标⾸先需要确 定进⾏ 双标 的 2 个⼀抗的种属来源 。如果来源不同 , 双标可以 同 时进⾏[1]

  1. The following day, the sections were rewarmed at room temperature for 1 h and washed three times with PBS for 5 min. [3]

  2. Secondary antibodies（⼆抗） [cat. no. ta130022; fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG, 1:64【最适浓度？】; and ta130012, Cy3-conjugated goat anti-mouse IgG, 1:50【最适 浓度？】；] were added and incubated away from light, at room temperature for 4 h, followed by washing in the dark with PBS for 5 min three times. Nuclear staining was performed with DAPI (OriGene Technologies, Inc.) for 5 min, and sections were washed in the dark with PBS for 5 min three times. [3]

fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG-荧光素异硫氰酸酯(FITC)结合 ⼭⽺抗兔IgG Cy3-conjugated goat anti-mouse IgG-cy3标记的⼭⽺抗小⿏IgG DAPI-脒基苯基吲哚(diamidino-phenyl-indole)

  1. 光源⽤激光器分别激发不同波⻓的荧光，Tissues were observed and photographed under a fluorescence microscope at magnification, x400 .[3]

    Discussion

在荧光染⾊的过程中。必须使⽤能激发出不同颜⾊的荧光物质来标 记第⼆抗体。常⽤的异硫氰酸荧光素(FITC)可以激发出绿荧光,Texas red和cy3激发红荧光。[1] 荧光产物需要⽤荧光显微镜在暗室检测 , 但检 查时间以每次 1 〜 2 h 为宜,超过2 h ,超⾼压汞灯发光强度逐渐下降 , 荧光减弱; 另外,标本受紫外线照 射 3 〜 5 min,荧光也会明显减弱,所以观察结果时 间最⻓不超过 3 h , 时间 久⻓ , 荧光染⾊逐渐减弱 , 因此荧光显微镜摄影 技术对于记录荧光图像⼗分重要 。标本染⾊后应⽴即集中观察并挑 选视野拍照。其拍照⽅法与普 通显微镜拍照技术基本相同 , 只是需要采⽤⾼速感 光胶⽚如 ASA 值 200 以上或 24 ℃以上的 ; 且因紫外 线对荧光淬灭作⽤⼤,所以曝光速度不能太慢 , ⼀般采⽤全⾃动显微摄影系统装置迅速曝光 。 对荧光染 ⾊结果可⽤图像分析仪进⾏半定量分析 。[2]

Reference

[1]路菊,孙玮,陈德英. 免疫荧光双重染⾊的激光共聚焦显微镜样品制备及观察[J]. 免疫学杂志,2007(03):344-345+350.

[2]王瑞,王航. 免疫荧光双标技术对神经递质共存现象的研究[J]. ⼭西医科⼤学学报,1999(04):382.

[3]Qiao L, Fu J, Xue X, et al. Neuronalinjury and roles of apoptosis and autophagy in a neonatal rat model ofhypoxia-ischemia-induced periventricular leukomalacia. Mol Med Rep.2018;17(4):5940-5949.