Fig 1.
Concept for retrospective studies/diagnostics using tissue lithography.
(a) Proposed strategy towards retrospective analysis of archival tissue sections. Microscale dewaxing can be performed on selected areas, enabling subsequent IHC staining of specific areas. The sample can then be stored again in the biobank/pathology archive for future analysis. (b) Tissue lithography on FFPE tissue sections using a microfluidic probe.
Fig 2.
Spatial resolution and patterning capabilities of tissue lithography on FFPE samples.
(a) Tissue lithography on a tonsil tissue microarray with chromogenic detection of CD45 proteins using diaminobenzidine. Partial staining of TMA cores highlights the protective effect of the remaining paraffin layer. (b) Large-scale tissue lithography on a tonsil tissue section.
Fig 3.
Multiplexed fluorescence-based IHC using tissue lithography.
(a) Schematic of the multiple dewaxing and staining steps performed. (b) Overlay image of two fluorescence channels showing multiplexed immunostaining of β-actin (red channel) and CD45 (green channel). (c) Overlay image of three fluorescence channels showing microscale partial staining of a TMA core. The tissue microarray was mounted using DAPI (blue channel) to highlight the non-stained areas of the sample.
Fig 4.
Micrometer-scale co-localization of IHC stains.
(a) Schematic of the workflow for localized dewaxing and antibody incubation with the MFP. (b) Overlay image of bright-field and fluorescence (red) images of a breast cancer tissue microarray. Half of the core was dewaxed, and three footprints were incubated with antibodies. (c,d) 20× and 40× images of local HER2-staining (membrane, red channel) and p53 (nucleus, green channel).
Fig 5.
Multi-core staining over multiple days on a single tissue microarray.
(a) Schematic workflow of the staining procedure over four days. After each staining step, the sample is stored in the repository, and retrieved on the next day. (b) Bright-field image of the four cores processed during the experiments. The image was taken on day 1 of the experiment, on which core 1 had been dewaxed (darker) and core 2, 3 and 4 were still protected with paraffin. (c) Fluorescence images of the stained cores after dewaxing and staining procedure. Specific HER2 staining is visible on cores 1, 2 and 3, whereas core 4 shows a negative/non-specific stain.
Fig 6.
MFP head with multiple inlets and integrated mixer for continuous tissue rehydration.
(a) Photograph and close-up SEM images of microfabricated MFP head. (b) Ethanol concentration measured in the flow confinement using fluorescence. A continuous gradient of ethanol to water can be generated to locally rehydrate a sample.
Fig 7.
Comparison of on-bench and MFP-based protocol for immunostaining of FFPE samples.