Protocol for tissue processing and paraffin embedding of mouse brains following ex vivo MRI

Abstract

Combining histology and ex vivo MRI from the same mouse brain is a powerful way to study brain microstructure. Mouse brains prepared for ex vivo MRI are often kept in storage solution for months, potentially becoming brittle and showing reduced antigenicity. Here, we describe a protocol for mouse brain dissection, tissue processing, paraffin embedding, sectioning, and staining. We then detail registration of histology to ex vivo MRI data from the same sample and extraction of quantitative histological measurements.

Keywords: Microscopy; Model Organisms; Neuroscience.

Graphical abstract

Figure 1

Image of the custom sample holder for ex vivo imaging (A) 3D printed sample holder. (B) The mouse skull is placed on the sample holder using the tooth notch. The holder is inserted into a 15 mL tube and filled with Fluorinert.

Figure 2

Photos demonstrating the procedure of dissecting the mouse brain (A) Example of mouse brain within the skull. (B‒D) Removing the brain from the skull. (E) Example of brain with the skull removed. (F) Mouse brain within the brain matrix. (G and H) Using a brain matrix to slice the brain. G and H show positioning for sagittal and coronal slicing respectively.

Figure 3

Photos demonstrating the embedding process for multibrain blocks (A) A silicone mold is used to ensure the block size will be suitable for standard 25 mm × 75 mm slides. (B) A printed guide is inserted into the embedding mold, (C) then a glass coverslip with double tape on the top side is placed on top of the printed guide. (D) The guide is used to assist placement of the brains within the molten wax, the coverslip allows to keep all brains on the same plane, and the double tape keeps the brains in place. (E) After the wax has set, the silicone mold, printed guide and coverslip with double tape can be easily removed. (F) Example of a multibrain block with brains positioned for sagittal sections.

Figure 4

Microtome workflow (A) Soak the block in Mollifex followed by (B) ice water. (C) Cut sections. (D) The section is floated on a warm water bath until smooth and then (E) placed on a glass slide. (F) Sections are allowed to dry upright at 18°C–22°C.

Figure 6

Photos of coverplates and slide rack used during immunohistochemistry (A) Coverplate. (B) Immerse the coverplate into water ensuring there are no bubbles on the surface of the coverplate then place the slide on top of the coverplate with the tissue facing the coverplate. Lift the coverplate/slide out of the water while squeezing slightly to prevent water leaking then insert the coverplate with slide into the rack (C).

Figure 5

Example staining strategy (A) For a single region of interest, we acquired neighboring sections with different stains, such as PLP, Neurofilament (NF), NeuN, CD68 and Ferritin close to the midline. (B) For multiple regions of interest, we acquired three different stains, PLP, NF, Brevican, per region of interest.

Figure 7

MRI-histology registration chain and examples of outcomes Framework for histology-MRI registration and extraction of quantitative histology metrics. (A) Multi-modal MRI to multi-stain histology registration workflow. Pydpiper is used for the standard-space registration and atlas-based segmentation of the dMRI data. FSL-FLIRT is used to co-register the multi-modal MRI data. TIRL is used for the MRI-histology and histology-histology registration. (B) Example MRI-histology (PLP) registration, red contour line represents the white matter boundary. (C) Example Multi-modal MRI registration result. R2∗ is registered to T2-weighted MRI, and FA (Fractional anisotropy) is registered to T2-weighted MRI. (Note FA was calculated using the diffusion tensor model). (D) Example histology-histology registration result. Neurofilament (NF) is registered to the PLP section, and Brevican is registered to the PLP section for the same sample. (E) PLP slice (coronal) displayed in 3D MRI space (represented by sagittal slice). (F) Quantitative histology metrics obtained from PLP: Structure tensor imaging and Stain Area Fraction map. The color wheel represents the fiber orientation. Red contour represents the white matter tract boundary. The white matter mask was manually segmented in standard diffusion-weighted MRI space with a visual aid from an atlas-based segmentation output and a tract skeleton (G) Example of a failed registration of an overprocessed sample which has shrinkage and folds.

Figure 8

Representative images comparing outcomes of the optimized and standard protocols (A and B) Representative images of the optimized protocol after sectioning (A) and after PLP staining with hematoxylin counterstaining (B). (C and D) Representative images of the standard protocol after sectioning (C) and after PLP staining with hematoxylin counterstaining (D). The representative images were sectioned and stained by the same operator. Folds (black arrowhead). Tears (white arrowhead). Scale bar is 2 mm.

Figure 9

Expected outcome Representative images of Brevican (A, enlarged in B), CD68 (C, enlarged in D), Ferritin (E, enlarged in F), NeuN (G, enlarged in H), Neurofilament (I, enlarged in J) and PLP (K, enlarged in L) staining. Neurofilament (NF). Perineuronal nets (black arrowhead) and macrophages/microglia (white arrowhead). Scale bar is 50 μm. Red box indicates location of higher magnification panel.