Biomed Opt Express. 2017 Jul 10;8(8):3583-3596.

Plastic embedding immunolabeled large-volume samples for three-dimensional high-resolution imaging

Yadong Gang1,2,5, Xiuli Liu1,2,5, Xiaojun Wang1,2,*, Qi Zhang1,2, Hongfu Zhou1,2, Ruixi Chen1,2, Ling Liu1,2, Yao Jia1,2, Fangfang Yin1,2, Gong Rao1,2, Jiadong Chen3,4, Shaoqun Zeng1,2.

1 Britton Chance Center for Biomedical Photonics, School of Engineering Sciences, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China

2 MOE Key Laboratory for Biomedical Photonics, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan, Hubei 430074, China

3 Department of Cell Biology and Program in Molecular Cell Biology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China

4 Second Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang 310058, China

5 These authors contributed equally to this work

* Correspondence should be addressed to Xiaojun Wang, Email:



High-resolution three-dimensional biomolecule distribution information of large samples is essential to understanding their biological structure and function. Here, we proposed a method combining large sample resin embedding with iDISCO immunofluorescence staining to acquire the profile of biomolecules with high spatial resolution. We evaluated the compatibility of plastic embedding with an iDISCO staining technique and found that the fluorophores and the neuronal fine structures could be well preserved in the Lowicryl HM20 resin, and that numerous antibodies and fluorescent tracers worked well upon Lowicryl HM20 resin embedding. Further, using fluorescence MicroOptical sectioning tomography (fMOST) technology combined with ultra-thin slicing and imaging, we were able to image the immunolabeled large-volume tissues with high resolution.



The development of diverse large-volume brain imaging techniques greatly enhanced our study on neuroscience. Recently, the appearance of various tissue clearing methods enabled the realization of immunofluorescence labeling and imaging of specific protein distributions and subcellular structures in intact tissue (Alanentalo et al., 2007; Chung et al., 2013; Renier et al., 2014). For imaging, although current methods, like light-sheet and serial two-photon microscopy methods, have showed their abilities in capturing large-volume datasets (Economo et al., 2016; Renier et al., 2014; Silvestri et al., 2013; Wilt et al., 2009), an optimal method that can realize coinstantaneous high speed and high resolution on immunostaining labeled samples is still under pursuing (Silvestri et al., 2013; Wilt et al., 2009). fMOST technique basing on serial ultra-thin slicing and imaging of resin embedded sample is an alternative way which may exert powerful strength to this goal (Gong et al., 2013; Li et al., 2010; Zhang et al., 2017; Zheng et al., 2013). Based on the reactivatable feature of plastic embedded fluorescent proteins, recently developed diverse fMOST systems had showed their power in detecting GFP/YFP labeled large samples (Gong et al., 2016; Gong et al., 2013; Xiong et al., 2014; Yang et al., 2015; Zhang et al., 2017; Zheng et al., 2013). But, if fMOST techniques are also applicable for imaging immunofluorescence or neural tracer labeled sample is still unknown. One of the crucial problem is how to preserve the state of immunostaining process and its fluorescent signals in resin embedding samples.


Our study first examined how various fluorescent probes behave after the embedding of different commonly used resins (Figure 1). Through testing on embedding TH-antibody immunostained brain slice samples by using different second antibodies with different probes, we found that Lowicryl HM20 and GMA are both good candidates for embedding immunofluorescence labeled samples (Figure 1c).


Since Lowicryl HM20 resin shows better ultra-thin slicing properties, we then did systematic testing on multiple antibodies, second antibody probes and fluorescent tracers on this resin. We found that the state of immunostaining process by antibodies and the fluorescence of probes are all preserved well after the Lowicryl HM20 resin embedding. The fine structures of neurites (anti-genetic labeled or viral labeled weak expressed fluorescent proteins, such as Thy1-GFP-M mice or PRV-DsRed labeled samples), vasculatures (Lectin-DyLight 594), and the distribution of endogenous PSD95 proteins or parvalbumin (PV) can be easily detected (Figure 2a-i).


Furthermore, We also combined the iDISCO (Renier et al., 2014) staining technique with fMOST imaging system (Yang et al., 2015) to study how our resin embedding works on imaging large-volume anti-TH mouse brain blocks. Our results presented the reliability of using our study to capture immunofluorescence labeled structures in large-volume biological tissue at high resolution (Figure 2j-l).



We acknowledge the support from Program 973 (2015CB755603). 



Figure 1. Fluorescence preservation of immunostained brain tissue before and after Lowicryl HM20 resin embedding. (a) Images of immunofluorescence stained brain tissue before and after HM20 resin embedding. A mouse brain tissue slice was immunofluorescence-labeled by primary antibodies (anti-tyrosine hydroxylase) and various secondary antibodies (CF488, CF568, CY3, or CY5-conjugated secondary antibodies). All images were recorded at a 0.42×0.42×1 μm3 voxel size on the same confocal microscope (ZEISS, 780) with the same configuration at room temperature. (b) Absorption spectra of different dyes (CF488, CF568, CY3, and CY5) in PBS buffer and Lowicryl HM20 resin polymer. (c) Comparison of the fluorescence change of immunofluorescence stained brain tissue in different resins (n = 4 independent samples). Data are shown as means ± SD. Scale bar: 50 μm.



Figure 2. Feasibility of using Lowicryl HM20 resin embedding to image immunofluorescence labeled samples with various antibodies and fluorescent probes. (a) Immunolabeling of tdTomato-labeled mouse cortex from ChAT-cre::Rosa26lsl-tdTomato transgenic mice. (b) TH-immunolabeled thalamic neurons in C57 mouse brain tissue. (c-d) Fluorescence images of neurons labeled by PRV-GFP virus; slices were imaged before immunofluorescent labeling (c), and after resin embedding on immunofluorescent labeling (d). (eh) Immunolabeled mouse brain slices by cFos (e), parvalbumin (f), PSD-95 (g), and cholera toxin beta (h) separately. (i) Lectin-DyLight 594 labeled vasculature. (I-II) Enlarged images. (j-l) fMOST imaging of Lowicryl HM20 resin embedded iDISCO immunofluorescence-stained large volume mouse brain block (Anti-TH). (k) and (l) are high resolution volume views enlarged from the boxes in (j). TH-positive neuron soma (l) and axon fibers (k) were shown respectively. Images were acquired by successive high-resolution stage-scanning microscopy at a 0.16 × 0.16 × 1.00 μm3 voxel size. Volume sizes: (i), 6200 × 5000 × 1200 μm3; (k), 480 × 480 × 380 μm3; (l), 480 × 480 × 510 μm3.




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