smFISH data of IEG expression in the dorsal striatum after acute, repeated, and challenge cocaine exposures

smFISH protocol was performed on14 mm tissue sections using the RNAscope Multiplex Fluorescent Reagent kit (Advanced Cell Diagnostics) according to the RNAscope Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue and the RNAscope Fluorescent Multiplex Kit User Manual (Advanced Cell Diagnostics). Image acquisition was performed using a Hermes high-definition cell-imaging system with 10 0.4 NA and 40 0.75 NA objectives. Five Z-stack images were captured for each of four channels – 475/28 nm (FITC), 549/15 nm (TRITC), 648/20 nm (Cy5), and 390/18 nm (DAPI). Image processing was performed using ImageJ software. Maximum-intensity images for each channel were obtained using Maximum Intensity Z-projection. All channels were subsequently merged, and the dorsal striatum region was manually cropped from these merged images according to the Franklin and Paxinos Mouse brain atlas, Third edition. Quantification of RNA expression from images was done using the CellProfiler (McQuin et al., 2018) speckle counting pipeline. A detailed protocol is available in Gonzales et al., 2020.本研究针对14 mm组织切片执行单分子荧光原位杂交（smFISH）实验方案，所用试剂为RNAscope多重荧光试剂试剂盒（Advanced Cell Diagnostics），操作严格遵循《RNAscope新鲜冷冻组织样本制备与预处理指南》及《RNAscope荧光多重试剂盒用户手册》（Advanced Cell Diagnostics）。图像采集采用Hermes高清细胞成像系统，搭配10×/0.4 NA及40×/0.75 NA物镜。针对4个成像通道——475/28 nm（FITC）、549/15 nm（TRITC）、648/20 nm（Cy5）及390/18 nm（DAPI），每个通道均采集5张Z-stack图像。图像处理通过ImageJ软件完成：先利用最大强度Z投影（Maximum Intensity Z-projection）提取各通道的最大强度图像，再将所有通道图像进行融合；随后参照《富兰克林与帕西诺小鼠脑图谱（第三版）》，从融合图像中手动裁剪出背侧纹状体区域。图像中RNA表达的定量分析采用CellProfiler（McQuin等，2018）的斑点计数流程完成，详细实验方案可参见Gonzales等（2020）的研究。