smFISH data of IEG expression in the dorsal striatum after acute, repeated, and challenge cocaine exposures
Description
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.
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Steps to reproduce
Single molecule fluorescence in-situ hybridization: A detailed protocol is available in (Gonzales et al., 2020). Briefly, smFISH protocol was performed on 14µm 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 10x 0.4NA and 40X 0.75NA objectives. 5 Z-stack images were captured for each of 4 channels-475/28 nm (FITC), 549/15 nm (TRITC), 648/20 nm (Cy5) & 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. smFISH analysis For the IEGs probes, selection for ‘robust-expressing’ cells was done as follows: We used the cocaine-naïve control data and after removing the non-expressing cells (cells expressing 0-1 puncta), the remaining cells were binned equally into three groups based on the per-cell expression levels, and the top 33% cells were defined ‘robust expressors’ or ‘suprathreshold cells’. Thus, cells qualified as ‘robust expressors’ for a given IEG if they expressed at least the following number of puncta per cell: Arc - 11, Egr2 - 6, Nr4a1- 12, Fos - 5. For Drd1 and Drd2 expression, a threshold of 8 puncta/cell was implemented (Gonzales et al., 2020) In order to identify the area with the highest density of IEG expressing cells in the striatum, we performed Two-Dimensional Kernel Density Estimation using the function ‘geom_density_2d’ in R [as in (Gonzales et al., 2020)]. This function estimates two-dimensional kernel density with an axis-aligned bivariate normal kernel, evaluated on a square grid, while displaying the result with contours. The regions of highest density, within which at least 20% of the cells are found, were selected. This process was performed independently for each one of the replicas and the selected contours plotted. Probes: Probe- Mm-Drd1a-C2 RNAscope® Cat No# 406491-C2 Probe- Mm-Drd1a-C3 RNAscope® Cat No# 406491-C3 Probe- Mm-Drd2-C2 RNAscope® Cat No# 406501-C2 Probe- Mm-Egr2 RNAscope® Cat No# 407871 Probe- Mm-Fos-C3 RNAscope® Cat No# 316921-C3 Probe- Mm-Arc-C3 RNAscope® Cat No# 316911-C3 Probe- Mm-Nr4a1-C2 RNAscope® Cat No# 423341-C2