Fig. 4: tDISCO resolves the boundaries and molecular profiles of proximal and distal astrocytes in sub-acute stroke.

A Schematic of the tDISCO system. tDISCO is comprised of an upright microscope equipped with a digital microfluidic (DMF) capable stage (grey, dashed inset), a fiber optic laser input and LED fluorescence capabilities. B Image shows three 2 mm tissue biopsies (i, ii, and iii, in dotted circles) from a cryosectioned tissue slice. Biopsies are mounted onto an tDISCO top-plate, fixed and stained. Cartoon of the biopsies in a brain section is shown on bottom right. Biorender was used to create the schematic. C Representative tDISCO-captured images of cortical brain tissue from which DAPI + NEUN+ neurons (red) and DAPI + GFAP+ astrocytes (green) were selected. Top image shows 20X field of view, scale bar is 100 µm. 100X images show NEUN+ (red dotted circles) and GFAP+ (green dotted circles) cells before (middle image) and after (bottom image) laser lysis. Scale bars are 10 µm. D Heatmap depicts differences in TPM normalized gene expression (columns) between NEUN+ and GFAP+ cells (rows). n = 4 GFAP+ and 4 NEUN+ cells from across 3 animals. E Heatmap depicts differences in LFQ normalized protein expression (columns) between NEUN+ and GFAP+ cells (rows). n = 3 GFAP+ and 3 NEUN+cells from across 3 animals. F Representative image of stroke-injured tissue stained for GFAP (green) from which GFAP+ astrocytes were selected. GFAP+ cells were isolated from three 200 µm “zones” (A through C) that spanned the d10 injury site (top image) and from a more distal zone (zone D, bottom image). Zone boundaries are shown by dotted lines, zone A is red, zone B is orange, zone C is light blue, and zone D is dark blue. The position of each selected astrocyte within the tissue is depicted by numbered circles. All scale bars are 100 µm. cc = corpus collosum, V = ventricle and ML = midline. G Transcriptome heatmap of numbered GFAP+ cells (1-22, bottom) from F (columns) isolated from zones A (red), B (orange), C (light blue) and D (dark blue). Gene expression is TPM normalized. Transcripts are grouped according to their localization in injury zone B (top), in every zone except injury zone B (second down), to distal zones C and D (third down) or lncRNA outside of zone B (fourth down). n = 22 cells from across 3 animals. H Proteome heatmap with numbered GFAP+ cells (23–32, bottom) from F (columns) isolated from zone B (orange) and zone D (dark blue). LFQ normalized intensity of proteins. Proteins are grouped according to expression within zone B (top) or in zone D (bottom). n = 10 cells from across 3 animals. Source data are provided as a Source Data file.