Supplementary Material for: An optimized and detailed step-by-step protocol for the analysis of neuronal morphology in golgi-stained fetal sheep brain

Antenatal brain development during the final trimester of human pregnancy is a time when mature neurons become increasingly complex in morphology, through axonal and dendritic outgrowth, dendritic branching, and synaptogenesis, together with myelin production. Characterizing neuronal morphological development over time is of interest to developmental neuroscience and provides the framework to measure grey matter pathology in pregnancy compromise. Neuronal microstructure can be assessed with Golgi staining, which selectively stains a small percentage (1-3%) of neurons and their entire dendritic arbor. Advanced imaging processing and analysis tools can then be employed to quantitate neuronal cytoarchitecture. Traditional Golgi staining protocols have been optimized and commercial kits are readily available offering improved speed and sensitivity of Golgi staining to produce consistent results. Golgi stained tissue is then visualized under light microscopy and image analysis may be completed with several software programs for morphological analysis of neurons, including freeware and commercial products. Each program requires optimization, whether semi-automated or automated, requiring different levels of investigator intervention and interpretation, which is a critical consideration for unbiased analysis. Detailed protocols for fetal ovine brain tissue are lacking and therefore, we provide a step-by-step workflow of computer software analysis for morphometric quantification of Golgi-stained neurons. Here, we utilized the commonly applied FD Rapid GolgiStain kit (FD NeuroTechnologies) on ovine fetal brains collected at 127 days (0.85) gestational age for the analysis of CA1 pyramidal neurons in the hippocampus. We describe the step-by-step protocol to retrieve neuronal morphometrics using Imaris imaging software to provide quantification of apical and basal dendrites for measures of dendrite length (μm), branch number, branch order and Sholl analysis (intersections over radius). We also detail software add-ons for data retrieval of dendritic spines including the number of spines, spine density and spine classification, which are critical indicators of synaptic function. The assessment of neuronal morphology in the developing brain using Rapid-Golgi and Imaris software is labour-intensive, particularly during the optimization period. The methodology described in this step-by-step description is novel, detailed, and aims to provide a reproducible, working protocol to quantify neuronal cytoarchitecture with simple descriptions that will save time for the next users of these commonly used techniques.

人类妊娠晚期三个月期间的产前脑发育阶段，成熟神经元会通过轴突与树突生长、树突分支形成以及突触发生，再加上髓鞘生成，在形态上愈发复杂。对神经元形态发育随时间变化的特征进行解析，是发育神经科学的研究热点之一，同时也为评估妊娠并发症中的灰质病理提供了分析框架。

神经元微观结构可通过高尔基染色（Golgi staining）进行评估，该技术可选择性染色1%-3%的神经元及其完整的树突分支丛。随后可借助先进的成像处理与分析工具，对神经元细胞结构进行定量分析。传统高尔基染色方案已得到优化，市售商用试剂盒可快速获取，能提升高尔基染色的速度与灵敏度，获得稳定一致的结果。

经高尔基染色的脑组织可在光学显微镜下成像，随后可通过多款软件完成图像分析以实现神经元形态学研究，这些软件涵盖免费开源工具与商业产品。无论半自动还是全自动分析，每款软件都需要进行参数优化，且对研究人员的干预与解读程度要求各异，这对于实现无偏倚分析而言是关键考量因素。

目前尚缺乏针对胎羊脑组织的详细操作方案，因此我们提供一套针对高尔基染色神经元形态计量定量分析的计算机软件分析分步工作流程。本研究采用市面常用的FD Rapid GolgiStain试剂盒（FD NeuroTechnologies公司），对妊娠127天（胎龄占比0.85）的胎羊脑组织进行染色，以分析海马CA1区锥体神经元。

我们详细描述了使用Imaris成像软件获取神经元形态计量参数的分步流程，可定量分析顶端树突与基底树突的相关指标，包括树突长度（单位：微米）、分支数量、分支阶数以及Sholl分析（半径相关的交叉点数量）。我们还详述了用于提取树突棘数据的软件插件功能，可获取树突棘数量、棘密度以及树突棘分类等数据，这些指标是突触功能的关键评价依据。

使用快速高尔基染色法与Imaris软件对发育中脑的神经元形态进行评估，工作量较大，尤其是在参数优化阶段。本分步指南所描述的方法具有创新性与细节完整性，旨在通过简洁易懂的说明，为后续使用这类常用技术的研究者节省时间，提供一套可重复的实用定量分析神经元细胞结构的方案。