Department of Pathology and Lab Medicine, Piscataway, NJ 08854, USA.
Exp Neurol. 2010 Oct;225(2):328-40. doi: 10.1016/j.expneurol.2010.07.004. Epub 2010 Jul 15.
The accurate and reliable counting of tyrosine hydroxylase positive (TH+) and tyrosine hydroxylase negative (TH-) neurons in the ventral midbrain is an important measure in studies related to Parkinson's disease and many other disorders associated with this region. Despite recent advancements, the use of stereology remains limited due to a variety of challenges for many users. We implemented a real-time fluorescence detection method and the use of an antibody to the neuron specific nuclear antigen (NeuN) to overcome some challenges for users. We found that the regional value for the two different cell types (TH+ and TH-) varied with the method of detection (chromogenic versus fluorescence) and with different nuclear markers (Nissl, DAPI, or NeuN). The number of both TH+ and TH- neurons was higher using fluorescence detection. The number of TH- neurons was higher using NeuN as a neuronal nuclear marker compared to DAPI. We identified 3 types of neuronal nuclei using NeuN staining characteristics. The method is applicable for mouse and rat. We describe a practical approach for epifluorescence-based counting of these two types of neurons that may offer significant advantages over existing methods for potential users.
准确可靠地计数腹侧中脑中的酪氨酸羟化酶阳性（TH+）和酪氨酸羟化酶阴性（TH-）神经元是与帕金森病和许多其他与该区域相关的疾病相关研究的重要衡量标准。尽管最近取得了进展，但由于许多用户面临各种挑战，立体学的应用仍然受到限制。我们实施了实时荧光检测方法，并使用神经元特异性核抗原（NeuN）抗体来克服用户的一些挑战。我们发现，两种不同细胞类型（TH+和 TH-）的区域值因检测方法（显色法与荧光法）和不同的核标记物（Nissl、DAPI 或 NeuN）而异。使用荧光检测时，TH+和 TH-神经元的数量更高。与 DAPI 相比，使用 NeuN 作为神经元核标记物时，TH-神经元的数量更高。我们使用 NeuN 染色特征鉴定了 3 种类型的神经元核。该方法适用于小鼠和大鼠。我们描述了一种基于荧光的这两种类型神经元计数的实用方法，对于潜在用户而言，它可能比现有方法具有显著优势。
