| Organotypic Explants of the Embryonic Rodent Hippocampus: An Accessible System for Transgenesis
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Author:
Date:
2018-03-20
[Abstract] This protocol describes the technique of ex-vivo electroporation to target embryonic hippocampal progenitors in an organotypic slice preparation. This technique allows gene perturbation for examining developmental processes in the embryonic hippocampus while retaining the environment and connectivity of the cells. Gene perturbation can include Cre-mediated recombination, RNAi-mediated knockdown, gene overexpression, or a combination of any of these. Ex-vivo electroporation can be performed at a wide range of embryonic stages, giving temporal control to the experimenter. Spatial control can be achieved more easily by preparing the brain in a Petri dish to target particular regions of the hippocampus. The electroporated explant cultures provide a highly tractable system ...
[摘要] 该协议描述了在器官切片制备中靶向胚胎海马祖细胞的体外电穿孔技术。 该技术允许基因摄动检查胚胎海马体的发育过程，同时保持细胞的环境和连接性。 基因扰动可以包括Cre介导的重组，RNAi介导的敲低，基因过表达或这些中任何的组合。 可以在广泛的胚胎阶段进行体外电穿孔，为实验者提供时间控制。 空间控制可以通过在皮氏培养皿中准备大脑来瞄准海马的特定区域来实现。 电穿孔的外植体培养物提供了用于研究包括祖细胞增殖，迁移和细胞命运获取在内的发育过程的高度易处理的系统。
【背景】由于其位于尾端的端脑，海马在可达性方面提出了挑战。胚胎海马甚至更难以接近，需要用子宫外科手术方法进行实验操作。器官切片文化规避了这个问题，同时保留了海马场细胞构筑学的许多方面，包括分子特征和连接性。尽管有描述来自啮齿类动物大脑的海马外植体产后培养的规程（Stoppini等人，1991; ...
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| Visualization of Intracellular Tyrosinase Activity in vitro
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Author:
Date:
2016-04-20
[Abstract] Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels result in loss of tyrosinase activity in melanosomes, commonly observed in albinism. Here, we describe a method to detect the intracellular activity of tyrosinase in mouse melanocytes. This protocol will visualize the active tyrosinase present in the intracellular vesicles or organelles including melanosomes.
[摘要] 黑素细胞在黑素体中产生黑色素，这些细胞器保护皮肤免受有害的紫外线。 酪氨酸酶是使用其底物氨基酸酪氨酸或L-DOPA（L-3,4-二羟基苯丙氨酸）引发颜料合成的关键铜酶蛋白酶。 此外，酪氨酸酶的活性与细胞色素沉着直接相关。 酪氨酸酶转运到黑素体中的缺陷或酶中的突变或降低的细胞内铜水平导致黑素体中酪氨酸酶活性的丧失，通常在白化病中观察到。 在这里，我们描述了一种方法来检测小鼠黑素细胞中酪氨酸酶的细胞内活性。 该协议将使存在于细胞内囊泡或细胞器（包括黑素体）中的活性酪氨酸酶可视化。
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