Embedding and Chemical Reactivation of Green Fluorescent Protein in the Whole Mouse Brain for Optical Micro-Imaging

Abstract

Resin embedding has been widely applied to fixing biological tissues for sectioning and imaging, but has long been regarded as incompatible with green fluorescent protein (GFP) labeled sample because it reduces fluorescence. Recently, it has been reported that resin-embedded GFP-labeled brain tissue can be imaged with high resolution. In this protocol, we describe an optimized protocol for resin embedding and chemical reactivation of fluorescent protein labeled mouse brain, we have used mice as experiment model, but the protocol should be applied to other species. This method involves whole brain embedding and chemical reactivation of the fluorescent signal in resin-embedded tissue. The whole brain embedding process takes a total of 7 days. The duration of chemical reactivation is ~2 min for penetrating 4 μm below the surface in the resin-embedded brain. This protocol provides an efficient way to prepare fluorescent protein labeled sample for high-resolution optical imaging. This kind of sample was demonstrated to be imaged by various optical micro-imaging methods. Fine structures labeled with GFP across a whole brain can be detected.

Keywords: chemical reactivation; embedding; fluorescent proteins; micro-imaging; whole mouse brain.

Figure 1

The principle reaction of chemical reactivation for fluorescence imaging. (A) Diagram of the course of GFP and chromophore changes in brain tissue during resin embedding and chemical reactivation. (B) The fluorescent state of nerve fiber tracts in the striatum region of resin-embedded thy1-GFPM mouse brain (left). (C) The fluorescent state reactivated by alkaline buffer solutions. (C) The red dashed line indicates the activation depth of alkaline buffer solutions. The z axis represents the alkaline buffer penetration depth. (D) Projection of the same z-stack from the imaging surface to the red dashed line in B and C. (E) Original fluorescence intensity distributions for the resin-embedded brain as a function of imaging depth. (F) Fluorescence intensity distributions after alkaline buffer activation for 2 min. (G) Fluorescence intensity distributions 1 μm below the surface of the resin-embedded brain over time. (E–G) The fluorescence intensity values are given as the mean ± SD (n = 6). Scale bars: (B,C), 20 × 20 × 50 μm³; (D), 2 μm.

Figure 2

Schematic diagram for the entire experimental procedure. (A) Schematic diagram illustrating the procedure for embedding whole mouse brains and chemically reactivating the quenched fluorescence. (B) Flowchart for the all steps of the protocol.

Figure 3

The influence of dehydrating temperature on CR-induced fluorescence recovery. (A) thy1-YFP H mouse brain was divided into the left hemisphere and the right hemisphere. The left and right hemispheres were dehydrated at 25 and 4°C, respectively, and then embedded in resin. After embedding, the same coronal plane area was revealed and immersed in 0.05 M Na₂CO₃ solution for 1 h. (A) Fluorescence brightness of the left hemisphere. (B) Fluorescence brightness of the right hemisphere. (A,B) Two regions of a single brain. The inset images in (A,B) show the same coronal plane area, and the green box indicates (A,B). (C) The normalized fluorescence intensities were comparable for the samples dehydrated at 25 and 4°C (mean ± SD; ^*p < 0.01 in two-tailed T-test; n = 32 each). Scale bars: (A,B), 100 μm; (A,B), green box, 0.5 mm.

Figure 4

The steps for polymerization. (A) Place the gelatin capsule in the hole of an aluminum base used for heat conduction when polymerizing samples. Then, transfer the brain into the gelatin capsule and immediately cover the lid of the gelatin capsule. (B) Place the sample in the oven. (C) At the end of polymerization, remove the sample from the oven. (D) Glue the sample onto a base plate.

Figure 5

CR in a 200 nm section from an HM20-embedded thy1-YFPH mouse brain. (A) The normal fluorescent state of YFP brain in a 200 nm section (left), the matching bright field image (center) and the overlay (right). (B) The YFP fluorescence reactivated by alkaline buffer solutions in a same section (left), the matching bright field image (center) and the overlay (right) are shown. Scale bars: (A,B) 20 μm.

Figure 6

Typical results obtained from CR of neurons and neurites in a resin-embedded thy1-GFPM mouse brain. (A,D,G,J) and (m) are maximum intensity projections of different coronal plane. (B,C), (E,F), (H,I), (K,L), (N,O) are corresponding magnification of regions indicated in (A,D,G,J,M), respectively. The projection thicknesses of (A,D,G,J,M) are 50 μm; (B,C,E,F,H,I,K,L,N,O) are 20 μm. Scale bars: (A,D,G,J,M) are 1 mm. (B,C,E,F,H,I,K,L,N,O) are 50 μm. All images were recorded on a commercial confocal microscope (Zeiss, LSM780).

Figure 7

Wide-field imaging of CR-enhanced resin-embedded thy1-GFPM mouse brain tissue. (A) Coronal plane of a mouse brain. (B) Layer V pyramidal neurons in the somatosensory cortex region. (C) Neural fiber tracts in the internal capsule and globus pallidus regions. Int, internal capsule; GP, globus pallidus. (D) Neurons and axonal collaterals in the amygdaloid nucleus. Scale bars: (A), 0.5 mm; (B–D), 100 μm.

Figure 8

Confocal imaging of neuronal morphology in CR-enhanced resin-embedded thy1-YFPH mouse brain. (A) A layer V pyramidal cell. (B) Spine on the apical dendrite of a pyramidal cell in the cortex region. (B) The white arrows and F indicate the filopodium. (C) Structure of axon branches and axon terminal boutons on layer 3/4. (D) The white arrows denote junction point structures. (D) Junction point structure of a dendrite and axon observed under light microscopy. All images were acquired at a 0.2 × 0.2 × 1 μm³ voxel size on a commercial confocal microscope (Zeiss, LSM710). Scale bars: (A), 50 μm; (B), (B) green box, (C), (C) green box, 5 μm; (D), 24 × 30 × 14 μm³.

Figure 9

Two-photon fMOST imaging of thy1-YFPH mouse brain. The inset image indicates the contours of the mouse brain labeled by blue region. Parts of the cerebral cortex are 3D reconstructed (the white box region inset) and enlarged. Scale bar: inset, 1 mm; the white box region, 1800 × 900 × 1500 μm³.