| Immunolabeling of Maize Meiocytes
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Author:
Date:
2020-04-05
[Abstract] This protocol describes a quick method for immunolocalization of meiotic proteins in maize. It includes a fixation step that allows for long-term storage of material and provides good preservation of chromatin structure.
[摘要] [摘要] 该协议描述了一种在玉米中进行减数分裂蛋白免疫定位的快速方法。它包括一个固定步骤，可以长期保存材料并提供良好的染色质结构保存。
[背景] 卵母细胞是生殖细胞，在其中发生同源染色体配对，突触和重组的专门过程，然后染色体分离以产生单倍体核。阐明减数分裂蛋白的定位对于理解这些过程至关重要。尽管此协议可能有多种变体，但我们发现微波加热步骤对于使其可靠地工作于玉米中的免疫定位至关重要。
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| Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration
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Author:
Date:
2020-03-20
[Abstract] In diseases such as glaucoma, the failure of retinal ganglion cell (RGC) neurons to survive or regenerate their optic nerve axons underlies partial and, in some cases, complete vision loss. Optic nerve crush (ONC) serves as a useful model not only of traumatic optic neuropathy but also of glaucomatous injury, as it similarly induces RGC cell death and degeneration. Intravitreal injection of adeno-associated virus serotype 2 (AAV2) has been shown to specifically and efficiently transduce RGCs in vivo and has thus been proposed as an effective means of gene delivery for the treatment of glaucoma. Indeed, we and others routinely use AAV2 to study the mechanisms that promote neuroprotection and axon regeneration in RGCs following ONC. Herein, we describe a step-by-step protocol to ...
[摘要] [摘要 ] 在青光眼等疾病中，视网膜神经节细胞（RGC）神经元无法存活或无法再生视神经轴突，这是部分视力丧失的原因，在某些情况下，甚至是完全的视力丧失。视神经挤压术（ONC）不仅可以作为创伤性视神经病变的一种有用模型，而且还可以作为青光眼损伤的有用模型，因为它类似地诱导RGC细胞死亡和变性。腺相关病毒血清型2（AAV2）的玻璃体内注射已被证明特别地和有效地转导视网膜神经节细胞在体内和已因而被提出作为基因递送用于治疗青光眼的治疗的有效手段。确实，我们和其他人常规使用AAV2来研究促进ONC 后RGC中神经保护和轴突再生的机制。本文中，我们描述了分步操作的方案，以测定AAV2介导的转导和ONC损伤后小鼠中RGC的存活和再生，包括1）玻璃体内注射AAV2病毒载体，2）视神经挤压，3）霍乱毒素B （CTB）标记再生轴突，4）视神经清除，5）视网膜平面免疫染色和6）定量RGC存活和再生。除了提供执行此协议所需的所有材料和程序详细信息之外，我们还强调了它比其他相似的已发表方法的优势，并提供了有用的技巧以确保其在任何现代实验室中都能如实复制。
[背景 ] 青光眼是世界范围内不可逆失明的主要原因，其特征是视网膜神经节细胞（RGCs）逐渐退化和丧失，这是构成连接视网膜与大脑的视神经的中央投射神经元（Quigley ，2011 ; Tham ...
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| Cell Surface Protein-protein Binding on COS-7 Cells
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Author:
Date:
2014-01-05
[Abstract] Examination of interactions between a transmembrane protein and a soluble protein by pull-down or immunoprecipitation assays can be tricky and complicated due to the detergent extraction of membrane proteins during the lysate preparation step. The choice and concentration of detergents must be determined empirically and the procedure can be burdensome. Here, we describe a simplified binding assay by expressing the membrane protein of interest in COS-7 cells and applying detergent-free solutions containing an extracellular protein to be tested. The binding is then examined by immunocytochemistry.
[摘要] 通过下拉或免疫沉淀测定法检查跨膜蛋白和可溶性蛋白质之间的相互作用可能是棘手和复杂的，因为在裂解物制备步骤期间膜蛋白的去污剂提取。 洗涤剂的选择和浓度必须根据经验确定，并且该方法可能是繁重的。 在这里，我们描述简化的绑定测定通过表达感兴趣的COS蛋白的COS 7细胞和应用含有待测试的细胞外蛋白的无洗涤剂溶液。 然后通过免疫细胞化学检查结合。
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