Immunolabeling-compatible PEGASOS tissue clearing for high-resolution whole mouse brain imaging - PubMed (original) (raw)
Immunolabeling-compatible PEGASOS tissue clearing for high-resolution whole mouse brain imaging
Pan Gao et al. Front Neural Circuits. 2024.
Abstract
Novel brain clearing methods revolutionize imaging by increasing visualization throughout the brain at high resolution. However, combining the standard tool of immunostaining targets of interest with clearing methods has lagged behind. We integrate whole-mount immunostaining with PEGASOS tissue clearing, referred to as iPEGASOS (immunostaining-compatible PEGASOS), to address the challenge of signal quenching during clearing processes. iPEGASOS effectively enhances molecular-genetically targeted fluorescent signals that are otherwise compromised during conventional clearing procedures. Additionally, we demonstrate the utility of iPEGASOS for visualizing neurochemical markers or viral labels to augment visualization that transgenic mouse lines cannot provide. Our study encompasses three distinct applications, each showcasing the versatility and efficacy of this approach. We employ whole-mount immunostaining to enhance molecular signals in transgenic reporter mouse lines to visualize the whole-brain spatial distribution of specific cellular populations. We also significantly improve the visualization of neural circuit connections by enhancing signals from viral tracers injected into the brain. Last, we show immunostaining without genetic markers to selectively label beta-amyloid deposits in a mouse model of Alzheimer's disease, facilitating the comprehensive whole-brain study of pathological features.
Keywords: Alzheimer’s disease; Light-Sheet; circuit tracing; tissue clearing; whole-mount immunostaining.
Copyright © 2024 Gao, Rivera, Lin, Holmes, Zhao and Xu.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
Figures
Figure 1
Specific single cell resolution immunostaining of whole brain cleared samples using iPEGASOS, visualized by Light-Sheet imaging of DAT-Cre;Ai9 mouse hemisphere. (A) iPEGASOS process. Top row: the state of the whole mouse brain at each stage of iPEGASOS. Bottom: in gold and green boxes, we depict each step, solution, and duration for the iPEGASOS processed whole mouse brain. (B) The autofluorescence background depicted in the top-volume view of the brain that is cleared but without immunostaining. The sample was imaged using 1.5x,0.37NA objective, Light-Sheet Microscopy, with the dorsal side facing up. (C) Lack of signal in a brain not incubated with primary and secondary antibodies shown in a horizontal optical plane in the midbrain region, showing very low background autofluorescence. (D) Antibody staining signal in a top volume view of a whole brain cleared following immunostaining using rabbit x DsRed antibody. Strong signal is seen in the midbrain region and olfactory bulb, indicating that the process allows immunolabeling of deep brain regions. (E) Specific DAT promoter driven tdTomato signal amplified with anti-DsRed antibody seen in a horizontal optical plane in midbrain region. (F,G) Higher magnified views of the white boxes at the midbrain region in (C,E) show low background for the no antibody control (F) and single-cell resolution signal for the antibody-treated brain (G), respectively. (H) Higher magnified views of a single optical plane at olfactory bulbs in panel C, showing low autofluorescence background. (I) Higher magnified views of a single optical plane at olfactory bulbs in panel E.
Figure 2
Single-cell resolution imaging can be achieved throughout the entire brain from the dorsal (Top imaging plane) to the ventral (bottom imaging plane) plane of the VIP-Cre;Ai9 mouse hemisphere. (A–F) Various depths of horizontal optical planes from a VIP-Cre; Ai9 mouse hemisphere cleared and immunostained using iPEGASOS and rabbit x DsRed antibody. It was acquired from a 1.5x Light-Sheet Microscopy. A to F are optical planes: 0.5, 1.1, 1.7, 2.3, 2.9 and 3.5 mm away from the dorsal part (top) of the brain. A, Anterior; P, Posterior; M, Medial; L, Lateral. (A1,A2–F1,F2) Single-cell resolution zoom-in of white-boxed regions in (A–F). (G) A digitally segmented cross-section of the brain features a 4.5 mm-tall column, organized from the dorsal (right) to ventral regions (left), including the cortical areas, hippocampus, thalamus, and midbrain.
Figure 3
3D rendering of the VIP-Cre;Ai9 mouse hemisphere showing dendritic-level resolution. (A) A rendered hemisphere viewed from the dorsal top. A, Anterior; P, Posterior; M, Medial; L, Lateral. (B) Maximum intensity projection created from 133 optical planes (400 μm thickness). (B1–B4) Top: Boxed regions in B are further enlarged to generate (B1–B4). Bottom: These images are the higher magnifications of the boxed region indicated on top.
Figure 4
iPEGASOS allows pseudorabies virus-based whole-brain neural circuit mapping. (A) The schematic drawing for the viral injection approach. (B) Starter cells are pointed with arrows in a single optical section (horizontal view). Cells with arrowheads are input neurons in RSCg. (B1) At a higher magnification, we zoom in on the starter cells, revealing the colocalization of DsRed from the rabies viral tracer and EGFP from AAV. (C) A 3D rendering provides a front view of a brain injected with a rabies viral tracer. The signal (DsRed) highlights input cells connecting to M2-projecting RSCg cells. The brain was imaged coronally. TH, Thalamus; DB, Diagonal band; RSC, Retrosplenial cortex. A, anterior; P, posterior; M, medial; L, lateral; D, dorsal; V, ventral. (C1–C4) Zooming in on the white boxed regions in panel C, we observe axon bundles (C1), single neurons and axons from thalamus input cells (C2), single-cell soma and neural processes from diagonal band input cells (C3), and axons projecting to the contralateral side of the injection site (C4). (D) A side view of the 3D brain provides a close-up, highlighting the regions of the RSC, TH, DB. (E) A single axon is traced from their origin in the thalamic soma to their endpoints in the RSC.
Figure 5
iPEGASOS captures the progression of beta-Amyloid accumulation in 5xFAD ranging from 6 m to 17 m of age. (A–E) Top views of 3D reconstructed brains are presented for different conditions: (A) 5xFAD, 17-month-old, male; (B) 5xFAD, 13-month-old, male; (C) 5xFAD, 11-month-old, male; (D) 5xFAD, 6-month-old, male; and (E) Wildtype, 8-month-old, female. The white signal corresponds to 6E10 antibody staining for beta-amyloid peptide. (A–E), (A1–E1), and (A2–E2) shared the same scale bar. The brains were imaged sagittally. A, anterior; P, posterior; M, medial; L, lateral; D, dorsal; V, ventral. (A1–E1) Side view of the corresponding brains in (A–E). (A2–E2) A maximum intensity projection is generated from 200 optical planes spanning 1.2 mm. Yellow arrows highlight key structures: frontal cortex, hypothalamus (HY), thalamus (TH), inferior colliculus (IC), and hindbrain (HB).(F) A single optical section is displayed from the brain of a 13-month-old male 5xFAD mouse. The yellow signal represents NeuN staining, while the white signal corresponds to 6E10 staining. (F1–F3) Higher magnifications at (F1) striatum, (F2) thalamus, and (F3) inferior colliculus.