Protocol for Xenium spatial transcriptomics studies using fixed frozen mouse brain sections

Abstract

Here, we present a protocol for Xenium spatial transcriptomics studies using fixed frozen mouse brain sections. We describe steps for intracardiac perfusion, cryosectioning, and floating section mounting of brain sections, which enable runs on the Xenium analyzer and data delivery. We demonstrate that, in addition to the 10× Genomics-validated formalin-fixed paraffin-embedded (FFPE) and fresh frozen sections, fixed frozen thin brain sections are compatible with the Xenium platform and provide excellent imaging and quantification results for spatially resolved gene expression. For complete details on the use and execution of this protocol, please refer to Ma et al.¹.

Keywords: RNA-seq; gene expression; molecular biology; neuroscience; systems biology.

Graphical abstract

Figure 1

Photomicrograph illustration of the steps involved in OCT embedding, cryosectioning, and free-floating section mounting onto Xenium slides under a dissection microscope

Figure 2

Fresh frozen brain cryosectioning and direct pickup of sections onto a Xenium slide, following the 10× Genomics fresh frozen protocol Note the unutilized space of the imaging area and excessive tissue curling.

Figure 3

(A) Tissue section layout on an experimental Xenium slide, which generated three datasets. (B) Overview of stained tissue on a Xenium slide. (C) Combined staining of cell nuclei, boundaries, and contents enables excellent cell segmentation. Tissue images and overlaying transcripts can be visualized using the Xenium Explorer browser.

Figure 4

(A) Xenium analyzer onboard analysis outputs allow for unsupervised clustering of the three datasets that are visualized in spatial domain. (B) The same three datasets visualized in UMAP embedding. (C) Supervised clustering and label transfer identified different cell type populations based on their marker genes.

Figure 5

Additional clustering and cell type annotation using the Scanpy pipeline (A) Unsupervised clustering resulted in 44 cell types across both hemisections. (B) Cell type label transfer was performed using the Allen Cortex reference dataset. (C–E) Cell cluster transitions and top marker genes for the annotated cell types are presented in the Sankey plot (C) and heatmaps (D and E).

Figure 6

The Sham/TBI dataset supports spatially resolved quantitative gene expression analysis (A) ROIs can be defined for selected cell type annotation. (B) UMAP visualization of cells in the selected ROIs. (C) Bar graph quantification of cell numbers in the selected ROIs. (D) Quantitative Gad1 gene expression using violin plots in "Pvalb" cell type. p = 0.38, Mann-Whitney U test. (E) Quantitative Gfap gene expression in the "Astro" cell type. p < 0.0001, Mann-Whitney U test.