Fig. 1
From: Nondestructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy
Workflow of transparent tissue tomography (T3) for whole core needle biopsies. a Tissue cylinders (cores) are collected using an automated 18 G needle (0.84 mm ID) core biopsy device and b cores are fixed with 2% paraformaldehyde and loaded into agarose wells. c Cores are stained overnight with cocktails of fluorescent primary antibodies. d After washing and fixing, cores are rendered transparent by equilibration with 100% (w/v) d-fructose, mounted between coverslips, and e scanned on one side by confocal microscopy in each fluorescent channel, then flipped and scanned on the other side. f The core is reconstructed by fusing half-cylinder images and g spatial analysis of the immune microenvironment is performed with Fiji macros. For secondary conventional IHC analysis, h imaged cores are washed free of d-fructose, fixed, and i submitted for FFPE processing and chromogenic IHC assay. Thereby, j correlated 2D and 3D data can be collected and integrated, providing a starting point for diagnostic, prognostic, and predictive testing for cancer immunotherapy
