Revealing intact neuronal circuitry in centimeter-sized formalin-fixed paraffin-embedded brain
- Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Taiwan
- Brain Research Center, National Tsing Hua University, Taiwan
- Institute of Biomedical Sciences, Academia Sinica, Taiwan
- Department of Physics, National Taiwan University, Taiwan
- Institute of Photonics Technologies, National Tsing Hua University, Taiwan
- Department of Neurosurgery, National Taiwan University Hospital, Taiwan
- Department of Pathology, National Taiwan University Hospital, Taiwan
- Institute of Molecular Biology, Academia Sinica, Taiwan
- Research Center for Applied Sciences, Academia Sinica, Taiwan
Figures
Figure 1 with 5 supplements
Development of the HIF-Clear pipeline.
(A) Comparison of DiD signal intensity in 2-mm-thick slices of undelipidated formaldehyde-fixed mouse brain, PFA-SDS mouse brain, and formalin-fixed paraffin-embedded (FFPE) mouse brain treated …
Figure 1—figure supplement 1
Examination of the antigen retrieval effect of SDS on formalin-fixed paraffin-embedded (FFPE) specimens.
(a) Light-sheet images of FFPE mouse brain hemisphere stained with tyrosine hydroxylase (TH) antibodies. The SDS treatment conditions are described above the images. Magnifications of dopaminergic …
Figure 1—figure supplement 2
Representative images of the results of DiD and Oil red O staining.
The delipidation effects of various detergent conditions were visualized and evaluated by lipophilic dye staining. Representative images of DiD (top row) and Oil red O (bottom row) staining are …
Figure 1—figure supplement 3
Repeated dewaxing test.
This test was designed to mimic experimental conditions where the sample is large and the optimal dewaxing time needs refinement. (a) Test procedure: a 2-mm-thick mouse brain block was fixed with 4% …
Figure 1—figure supplement 4
Repeated dewaxing does not influence antigenicity.
The brain block subjected to four rounds of dewaxing (as shown in Figure 1—figure supplement 3) was labeled with lectin to examine antigenicity. (a) Projection image of the entire block. (b) 3D …
Figure 1—figure supplement 5
Gross view of the mouse brain at different stages of the HIF-Clear protocol.
(a) 4% paraformaldehyde (PFA) fixation. (b) Formalin-fixed paraffin-embedded (FFPE) embedding. (c) Optimized antigen retrieval. (d) Refractive index (RI) matching. The FFPE mouse brain remain intact …
Figure 2
Validation of the HIF-Clear pipeline.
(A) Brain-wide tyrosine hydroxylase (TH) expression (left panel) and registration (right panel) in FFPE-HIF-Clear C57BL/6 mouse brains. Whole mouse brain images were acquired using light-sheet …
Figure 3 with 5 supplements
Application of HIF-Clear to long-term fixed mouse brain hemispheres.
Mouse brain hemispheres were fixed under various fixation conditions and then subjected to formalin-fixed paraffin-embedded (FFPE) processing and HIF-Clear. The specimens were stained with tyrosine …
Figure 3—figure supplement 1
Immunolabeled whole mouse brain without post-fixation exhibits faint or negative staining.
Light-sheet images of a formalin-fixed paraffin-embedded (FFPE) mouse brain stained with tyrosine hydroxylase (TH) and parvalbumin (PV) with (a, c, e) or without (b, d, f) post-immunolabeling …
Figure 3—figure supplement 2
Post-immunolabeling-fixed tyrosine hydroxylase (TH) signal remains after SDS stripping.
A 2-mm-thick formalin-fixed paraffin-embedded (FFPE) mouse brain specimen was stained with TH and then underwent post-immunolabeling fixation with 4% paraformaldehyde (PFA). Multi-point confocal …
Figure 3—figure supplement 3
Classic heat-induced epitope retrieval (HIER) facilitates relabeling after post-immunolabeling fixation.
A second round of staining, using lectin to label blood vessels, was performed on human brain tissues that had undergone an initial immunolabeling and post-fixation process. For the sample without …
Figure 3—figure supplement 4
Photobleaching in HIF-Clear+.
(a) A representative photobleaching apparatus. The 100 W projection lamp with an LED array (1), the multi-well plate containing refractive index (RI) matching solution and the sample (2), and the …
Figure 3—figure supplement 5
Examination of issue structural integrity and secondary antibody cross-reactivity of HIF-Clear+.
(a) Light-sheet images of a formalin-fixed paraffin-embedded (FFPE) mouse brain after each round of immunolabeling. 3D reconstruction images and magnification of the optical section in the center …
Figure 4 with 1 supplement
Multi-round immunostaining of FFPE-HIF-Clear whole mouse brain (HIF-Clear+).
Light-sheet microscopy imaging datasets of six-round immunolabeling performed on one whole FFPE mouse brain. Projection images of the horizontal (XY), sagittal (YZ), and transverse (XZ) views are …
Figure 4—video 1
Spatial distribution of the six biological markers shown in Figure 4.
Figure 5
Merged multichannel neuron circuitry image generated by HIF-Clear+.
(A) Merged multichannel image generated using the datasets from Figure 3. Optical sections from the horizontal (XY) and sagittal (YZ) perspectives are presented. For the transverse (XZ) views, …
Figure 6 with 1 supplement
Multi-round immunostaining of an FFPE-HIF-Clear human brain specimen.
Three-round immunolabeling was performed on an FFPE human brain specimen collected from a patient with cerebral hemorrhage. Images were acquired using multi-point confocal microscopy. (A) 3D …
Figure 6—figure supplement 1
Comparison of staining qualities of human and mouse brain tissues.
Multi-point confocal images of tyrosine hydroxylase (TH)- and lectin labeling in 1-mm-thick human and mouse brain tissues at depths of 100, 500, and 900 μm are shown.
Figure 7 with 4 supplements
Applications of HIF-Clear in disease models.
(A–F) HIF-Clear reveals cell–tumor relationships in an astrocytoma model. (A) Projection light-sheet image of an FFPE-HIF-Clear mouse brain with a GFP-expressing astrocytoma xenograft (ALTS1C1 …
Figure 7—figure supplement 1
Segmentation of dopaminergic regions and blood vessels in PFA-SDS traumatic brain injury (TBI) mouse brains.
(a) Whole-brain projection light-sheet image focusing on tyrosine hydroxylase (TH)-positive regions. (b) Frontal view of segmented striatum (dashed outline in a). Magenta: the injured side; cyan: …
Figure 7—figure supplement 2
Quantifications of FFPE-HIF-Clear and PFA-SDS samples result in identical statistical trends.
(a) Quantification of FFPE-HIF-Clear and PFA-SDS brains reveals identical inter-subgroup differences. n = 3 (three indicators: striatum volume, nigrostriatal fiber tract volume, substantia nigra …
Figure 7—video 1
Spatial relationships of the tumor and surrounding astrocytes in Figure 7A–E.
The segmented astrocytes are colored according to the cell-to-tumor distance code.
Figure 7—video 2
3D visualization of the segmented blood vessels shown in Figure 7L.
Figure 8 with 1 supplement
Expansion microscopy on an FFPE-HIF-Clear human brain specimen.
(A) Gross views of expansion of a 1 mm FFPE specimen. (B) Fluorescence images of an FFPE human brain pre- (top row) and post-expansion (bottom row). Left: merged images of multiplexed staining; …
Figure 8—figure supplement 1
Effects of various antigen retrieval conditions on formalin-fixed paraffin-embedded (FFPE) mouse brains.
Light-sheet images of an FFPE mouse brain hemisphere stained with tyrosine hydroxylase (TH) antibodies to evaluate the effects of different antigen retrieval methods. (a) The position of the Y-Z …
Additional files
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Supplementary file 1
The antibodies used in the study.
- https://cdn.elifesciences.org/articles/93212/elife-93212-supp1-v1.xlsx
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Supplementary file 2
Abbreviations of brain regions mentioned in Figure 2B.
- https://cdn.elifesciences.org/articles/93212/elife-93212-supp2-v1.xlsx
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Supplementary file 3
The antibody conditions in the multi-round immunolabeling shown in Figure 3.
- https://cdn.elifesciences.org/articles/93212/elife-93212-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/93212/elife-93212-mdarchecklist1-v1.docx
