Buford Jacob, Wilkins Owen, Kolling Fred, Leach Steven D, Morhardt Duncan
Department of Surgery, Dartmouth Hitchcock Medical Center;
Dartmouth Cancer Center.
J Vis Exp. 2025 May 16(219). doi: 10.3791/67683.
Spatial transcriptomic techniques are a sophisticated tool in biomedical research to visualize spatially registered gene expression patterns. Imaging and analysis of multiple samples with spatial imaging platforms can be costly. Performing these tests over multiple experimental conditions, as seen in developmental studies, further increases costs. To reduce costs, this study sought to optimize the techniques and strategies of spatial transcriptomic specimen arrangement for developmental studies. Here, the study utilized zebrafish, which are a well-established developmental vertebrate model that is transparent during development, have ~70% genetic homology to humans, and a highly annotated genome ideal for transcriptomic analysis. Because of their small size, developing zebrafish also allows for compact placement of serial sections across several biological replicates. Herein, we report optimized fixation, cryosectioning, and reliable alignment of multiple fish samples within the imaging area of a multiplex in situ hybridization spatial imaging platform. With this method, zebrafish as young as 15 days post fertilization (dpf) from at least 4 different molds and up to 174 sections can be successfully cryosectioned, collected within the imaging area of 22 mm 10.5 mm (for an in situ spatial transcriptomic slide), and processed simultaneously. Based on section quality, sample alignment, and sample size per slide, this method in zebrafish optimizes output and per sample cost of spatial transcriptomic techniques.
空间转录组技术是生物医学研究中用于可视化空间定位基因表达模式的一种精密工具。使用空间成像平台对多个样本进行成像和分析成本高昂。在发育研究中，在多个实验条件下进行这些测试会进一步增加成本。为了降低成本，本研究旨在优化发育研究中空间转录组样本排列的技术和策略。在此，该研究利用了斑马鱼，斑马鱼是一种成熟的发育脊椎动物模型，在发育过程中是透明的，与人类有~70%的基因同源性，并且有一个非常适合转录组分析的高度注释基因组。由于斑马鱼体型小，发育中的斑马鱼还允许在多个生物学重复样本中紧凑地放置连续切片。在此，我们报告了在多重原位杂交空间成像平台的成像区域内对多个鱼类样本进行优化固定、冷冻切片以及可靠对齐的方法。使用这种方法，受精后15天（dpf）的斑马鱼，至少来自4个不同模型，最多174个切片，可以成功地进行冷冻切片，收集在22毫米×10.5毫米的成像区域内（用于原位空间转录组玻片），并同时进行处理。基于切片质量、样本对齐以及每张玻片上的样本大小，斑马鱼中的这种方法优化了空间转录组技术的产出和每个样本的成本。
