Ba 2001

The frozen skin biopsy specimens were embedded in optimal cutting temperature compound and six µm thin sections were prepared in a microtome-cryostat, two sections per glass slide. The tissue architecture and degree of cell infiltrates were evaluated by hematoxylin and eosin staining. For the evaluation of T cell markers, the sections were acetone fixed and stained using the avidin–biotin complex method (Vectastain Elite ABC-kit, Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions with the following primary antibodies: anti-CLA (rat IgM, clone: HECA-452), anti-CD4 (mouse IgG₁, clone: SK3), anti-CD3 (mouse IgG₁, clone: SK7), all from BD Pharmingen (San Diego, CA, USA). Rat IgM (clone R4-22, BD Pharmingen) and mouse IgG₁ (clone MG1-45, Biolegend) were included in each staining as isotype controls. Biotinylated antibodies targeting rat IgM (clone G53-238, BD Pharmingen) and mouse IgG₁ (BA-2001, Vector Laboratories) were used as secondary antibodies. The sections were counterstained with hematoxylin.

Fixed hemispheres and spinal cords were cut on a cryotome (Leica, Nussloch, Germany) at 40 μm thickness of the sections. Immunohistochemistry on free-floating sections was performed using standard protocols and the following antibodies: anti-aggregated α-synuclein (5G4, Linaris, Germany), anti-phosphorylated α-synuclein (pS129, ab51253, Abcam, United Kingdom), anti-Iba1 (ab108539, Abcam, United Kingdom), anti-tyrosine hydroxylase (TH, AB152, Millipore, Germany), anti-CD68 (MCA1957GA, Serotec, United Kingdom), biotinylated anti-mouse, anti-rat and anti-rabbit IgG (respectively, BA-1000, BA-9400 and BA-2001, Vector Laboratories, United States). The reaction was enhanced with ABC Elite kit (PK-6100, Vector Laboratories, United States) and visualized with 3, 3′-diaminobenzidine. Cresyl violet was used for counterstaining of sections immunostained with anti-Iba1 antibody. For co-localization analysis, immunofluorescence for pS129 and LC3b (Cell Signaling, Leiden, Netherlands) followed by Alexa 488- and Alexa 594-conjugated secondary antibodies (Thermo Fisher Scientific, Rockford, IL, United States) was performed.

Kidneys and ventricle tissue fixed in formalin were sent to the Children’s Research Institute Histology Core at the Medical College of Wisconsin, where they were processed, paraffin embedded, sectioned (4 µM), mounted on slides, and Masson’s trichrome stained. Trichrome stained kidney sections were then imaged at 20 × using an E-400 microscope (Nikon) and SPOT Insight V5.1 digital camera (Diagnostic Instruments). Ventricle sections were scanned using a Super Coolscan 9000 (Nikon). The area of extracellular matrix protein containing (i.e. fibrotic; blue) tissue was quantified using Metamorph software and expressed as fibrosis percentage of total tissue area^{40 (link)}.
Immunohistochemistry was performed for detection of CD177 antigen, as previously described^{38 (link)}. CD177 antibody (sc-376329; Santa Cruz Biotechnology) was used at a 1:50 dilution and a rat adsorbed anti-mouse secondary antibody (#BA-2001; Vector Laboratories) was used at a 1:200 dilution. A secondary control slide (no primary antibody; 1:200 secondary antibody) slide was run in parallel with no staining observed.

We used our published procedure (28 (link), 47 (link)) to perform immunohistochemistry. Two- to 3-month-old female mice were anesthetized and perfused with physiological saline and then with 4% paraformaldehyde. The brains were kept in the fixative overnight at 4°C. Then, the mouse brains were cryoprotected in 20% glycerol in 0.1 M phosphate buffer overnight at 4°C. The mouse brains were cut into 40-μm thick sections. Free-floating sections were stained with a rabbit monoclonal anti-calretinin antibody (1:100, MA5–14540, Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) followed by a biotinylated goat anti-rabbit IgG (1:500, BA-2001, Vector Laboratories, Inc., Newark, CA, USA) and an avidin-biotin complex (Vectastain Elite, ABC kit peroxidase, Standard, Vector Laboratories, Inc, Newark, CA), using our standard 3,3′-diaminobenzidine staining procedure. Sections were examined using a Keyence BZ-X800 microscope (Keyence, Osaka, Japan). Each section containing the amygdalopiriform transition area was randomly chosen from both hemispheres. The calretinin-positive neuropil was delineated within the anteroposterior extent of the region (Bregma from −2.46 mm to −3.64 mm from bregma). The extent of stained neuropil was measured using Keyence analytical software in up to seven sections per mouse. The average of each mouse was used for analysis.

Four weeks after pMCAO, NS and S pigs were euthanized by IV pentobarbital (1 mL/4.5 kg) injection. Brains from both groups were removed and immersed in 10% buffered formalin (Millipore Sigma). Next, brains were sectioned coronally using a pig brain slicer (Zivic Instruments, Pittsburgh, PA). Right (ipsilateral to pMCAO) hemisphere sections were formalin-fixed, embedded in paraffin, and sectioned for immunohistochemistry (Leica RM2255, Germany). Following an enzyme block in 3% H₂O₂ for 5 min and Power Block (BioGenex) for 5 min, 4 μM thick sections were incubated with primary antibodies for 1 h on a Biocare Medical Nemesis 7200 Autostainer. Primary antibodies used were GFAP (mouse 1:4,000, Biogenex MU020-UC, Clone GA-5), IBA1 (rabbit 1:8,000, Wako, 019-19741), NeuN (guinea pig 1:3,000, Millipore Sigma, ABN90), FactorVIII (rabbit, Cell Marque, 250A-18), and DCX (rabbit, 1:4,000, Abcam, ab18723). Secondary antibodies used were Biotinylated goat anti-rabbit 1:100, Vector Labs, BA-1000 for IBA1, Biotinylated horse anti-mouse 1:100, Vector Labs, BA-2001 for GFAP, Biotinylated goat anti-guinea pig 1:100, Vector Labs, BA-7000 for NeuN, Biotinylated goat anti-rabbit 1:100, Vector Labs, BA-1000 for FactorVIII, and Biotinylated goat anti-rabbit 1:100, Vector Labs, BA-1000 for DCX. The substrate is DAB + Chromogen (12 min) from Biocare and all were counterstained with hematoxylin.