Rodent brain slicer matrix

After euthanasia by CO₂ asphyxiation, the brains were immediately extracted and kept ice-cold during dissection. The brains were sliced using a rodent brain slicer matrix (Zivic Instruments, Pittsburg, PA). Cortex samples were collected from 2 mm central coronal sections of each brain. RNA isolation was performed as described previously [2 (link)]. RNA samples were processed by the Institute for Genome Science at the University of Maryland School of Medicine using the nCounter custom-designed Nanostring gene panel (Nanostring Technologies, Seattle, WA, USA), which consisted of genes that are expressed predominantly by astrocytes (www.brainrnaseq.org). Only samples with an RNA integrity number RIN > 7.2 were used for Nanostring analysis. All data passed quality control, with no imaging, binding, positive control, or CodeSet Content Normalization flags. The analysis of data was performed using nSolver Analysis Software 4.0. Ten house-keeping genes (Xpnpep1, Lars, Tbp, Mto1, Csnk2a2, CCdc127, Fam104a, Aars, Tada2b, and Cnot10) were used for the normalization of gene expression.

Three days after stroke, the brains were removed and cleaved into four coronal sections with a 2.0-mm slice interval using a rodent brain slicer matrix (Zivic Instruments, Pittsburgh, PA), as we reported [7 (link), 18 (link)]. Sections were incubated in 2% 2,3,5-triphenyletrazolium chloride (TTC; #T8877, Sigma-Aldrich, St. Louis, MO). Infarct volume (percent of hemispheric volume) was determined by one blinded observer and corrected for edema using the NIH Image J program (Image J 1.37v; Wayne Rasband, available through NIH) as described previously.

After euthanasia by CO₂ asphyxiation, brains were immediately extracted and kept ice-cold during dissection. Brains were sliced using a rodent brain slicer matrix (Zivic Instruments). 3 mm central coronal sections of each brain were formalin-fixed and embedded in paraffin. 4 μm sections were produced using a Leica RM2235 microtome (Leica Biosystems), mounted on slides, and processed for immunohistochemistry.
Rehydrated slides were subjected to the procedure of epitope exposure that involved hydrated autoclaving at 121 °C for 20 min in trisodium citrate buffer, pH 6.0, with 0.05% Tween 20. All antibodies used in this study are listed in the Key Resources Table. Immunofluorescence detection was performed with AlexaFluor-488, AlexaFluor-546, or DyLight-350 labeled secondary antibodies. An autofluorescence eliminator (Sigma-Aldrich) was used according to the original protocol to reduce background fluorescence. Fluorescent images were collected using an inverted Nikon Eclipse TE2000-U microscope (Nikon Instruments Inc) equipped with an illumination system X-cite 120 (EXFO Photonics Solutions Inc) and a cooled 12 bit CoolSnap HQ CCD camera (Photometrics). Images were processed using WCIF ImageJ software (National Institute of Health). In triple staining images, DyLight-350 signal was artificially colored as white for better visualization.

Male C57BL/6J mice (2–5 months) were used as the experimental model and were obtained from Charles River and maintained under barrier-fed and specific pathogen-free conditions with a standard balanced diet and free access to water, at a temperature of 20–24 °C and humidity of 45 to 65%. Mice were humanely culled by a schedule-1 procedure in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986. All experiments were approved by the local ethics committee and UK home office.
The brain, heart, and aorta were promptly removed after culling and collected in cold physiological saline (artificial cerebral spinal fluid, ACSF; see the next section). According to Paxinos and Franklin’s Mouse in Stereotaxic Compacts—4th edition, the brain regions of the PAG and PMC in mice were dissected using a Rodent Brain Slicer Matrix (Zivic instruments, Pittsburgh, PA, USA) with 0.5 mm coronal slice intervals, under microscopic guidance. All areas were approximately 500 µm × 1000 µm to ensure that the specialized area was included (see Table 1). The muscular layer was removed from the aorta and the intima layer was used for the experiment. A small piece (approximately 5 mg) of myocardium was taken from the left ventricular apex.

Immediately following the final LMA test session, the mice were euthanized by cervical dislocation, and brains were removed and stored at -80°C until microdissection to obtain striatal tissue samples. Trunk blood samples from the BEC-Test cohort were also collected and centrifuged at 5,000 rpm for 10 min to obtain plasma, which was stored at -80°C until use. Brains from the Immuno-Home cohort were sliced into 2 mm sections using a rodent brain slicer matrix (Zivic Instruments, Pittsburgh, PA), and unilateral 1.5 mm tissue punches (Miltex, Inc., York, PA) from the left anterior-dorsal striatum at 1.5 to -0.5 mm anterior to bregma were taken and stored at -80°C until immunoassay. A unilateral punch was then homogenized for approximately 20 seconds in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 2mM EDTA, 1% triton X-100, 10% glycerol with Phosphatase Inhibitor Cocktail 2 P5726, Phosphatase Inhibitor Cocktail 3 P0044 and Protease Inhibitor P8340 from Sigma Aldrich, St. Louis, MO), then centrifuged at 4°C for 10 min at 10,000 x g, and the protein was quantified using a Pierce 660 nm protein assay using a bovine serum albumin standard (Thermo Scientific, Rockford, IL).