High-Throughput Strategy for Profiling Sequential Section With Multiplex Staining of Mouse Brain

Abstract

The brain modulates specific functions in its various regions. Understanding the organization of different cells in the whole brain is crucial for investigating brain functions. Previous studies have focused on several regions and have had difficulty analyzing serial tissue samples. In this study, we introduced a pipeline to acquire anatomical and histological information quickly and efficiently from serial sections. First, we developed a serial brain-slice-staining method to stain serial sections and obtained more than 98.5% of slices with high integrity. Subsequently, using the self-developed analysis software, we registered and quantified the signals of imaged sections to the Allen Mouse Brain Common Coordinate Framework, which is compatible with multimodal images and slant section planes. Finally, we validated the pipeline with immunostaining by analyzing the activity variance in the whole brain during acute stress in aging and young mice. By removing the problems resulting from repeated manual operations, this pipeline is widely applicable to serial brain slices from multiple samples in a rapid and convenient manner, which benefits to facilitate research in life sciences.

Keywords: high-throughput; mouse brain; multiplex staining; registration; serial sections.

FIGURE 1

An integrated pipeline for processing batch tissue sections. (A) The procedure of acquiring continuous batch slices. The samples are embedded and sectioned in agarose. (B) The staining process for batch sections. Using the fitting and separation of the 24-well plate, the staining plate may efficiently realize the rapid processing of batch slices. (C) Diagram of registration and signal recognition.

FIGURE 2

Multiple views of structural information based on immunostaining. (A) Maximum intensity projections of the representative coronal planes of immunofluorescence staining of the NeuN in 50 μm thickness in the whole mouse brain. The scale bar is 5 mm. (B) Coronal results of the NeuN antibody, MBP antibody, and FNB staining. The scale bar is 1 mm. (C) Partially enlarged view of the area is shown in subgraph B. The scale bar is 300 μm.

FIGURE 3

Brain registration procedures. (A) The procedure to get the corrected atlas section by rotating the coronal plane to the slant slice plane. (B) The registration principle of brain slice and atlas. (C) The results of batch brain slice registration from the same sample. (D) The results of batch brain slice registration from a different kind of sample. The scale bar is 1 mm.

FIGURE 4

c-Fos activation in young and aging mouse brains in response to forced swimming stress. (A) Three behavior postures of the mouse in FST, namely, immobility, swimming, and climbing. (B) The aging mice showed a significant reduction in the number and time of climbing in the FST. For all panels, n = 9 per group, * indicates false discovery group (FDR) q-value < 0.05, based on P-values from unpaired two-sample t-test comparing the young group to the aging group. (C) A large number of neuron signals appeared in many cortical and subcortical areas. (D) The analysis of c-Fos activation in multiple subcortical brain areas. For all panels, n = 4 per group, * indicates false discovery group (FDR) q-value < 0.05, ** indicates FDR q-value < 0.01, based on P-values from unpaired two-sample t-test comparing the young group to the aging group. Scatter points represent the c-Fos activation of each animal. Error bars, mean ± SEM. (E) c-Fos activation was significantly reduced in the PAG, PVT, VMH, and LS in aging mice. The scale bars are for (C) 2 mm and for (E) 200 μm.

FIGURE 5

Multicolor counterstaining indicates that PV and ChAT neurons in some brain areas participate in the neural circuit response in FST. (A) The location of the selected slices. (B) PV+ neurons in the PL, the primary motor cortex (MOp), and ChAT+ neurons in the horizontal limb of the diagonal band (HDB) are merged with c-Fos in FST. White arrowheads indicate c-Fos+/PV+, and c-Fos+/ChAT+ neurons. Scale bars: (A) 1 mm and (B) 100 μm.