Potassium hexacyanoferrate trihydrate

Right hemispheres were fixed in 4% paraformaldehyde (Boston BioProducts, Ashland, MA) at 4 °C, examined for surface CMH, and sectioned into 40-μm coronal sections using a vibratome (Technical Products International, Inc., St. Louis, MO). Every fourth, fifth, sixth, and seventh section was collected. Every sixth section was stained with H&E by Research Services Core offered by the Department of Pathology and Laboratory Medicine at UCI Medical Center to detect fresh (acute) CMH. Every seventh section was stained with Prussian blue (PB) to detect hemosiderin (a marker of sub-acute CMH) [16 (link)]. Briefly, sections were stained using 5% potassium hexacyanoferrate trihydrate (Sigma, St. Louis, MO) and 5% hydrochloric acid (Sigma, St. Louis, MO) for 30 min, rinsed in water and counterstained with Nuclear Fast Red (Sigma, St. Louis, MO), dehydrated, and cover slipped. Remaining sections were used for immunohistochemistry. CMH were counted at a × 20 magnification by a blinded observer, and digitized images were used to determine CMH size (μm²) and positive area (expressed as a percent of total area analyzed), by an observer blinded to the experiment using RGB CMH analyzer program and NIH Image J software 1.62, respectively [16 (link)].

The brains were sectioned into 20-μm or 40-µm coronal sections with a freezing microtome (Thermo Fisher Scientific, Cleveland, OH, USA). Every seventh section was collected for Prussian blue staining to detect hemosiderin (marker of CMH formation) performed by the Research Services Core (Department of Pathology and Laboratory Medicine at UCI Medical Center). Briefly, sections were stained with freshly prepared 5% potassium hexacyanoferrate trihydrate (Sigma-Aldrich, St. Louis, MO, USA) and 10% hydrochloric acid (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. After rinsing in water, sections were counterstained with nuclear fast red, dehydrated, and coverslipped. To quantify CMH number, CMH were detected and photographed at 20× magnification with a light microscope by a blinded observer. Whole slide images were scanned to quantify the total area of the brain section. The area of the section was analyzed by a blinded observer using National Institute of Health (NIH) ImageJ software 1.52. CMH number was then adjusted to total area of the brain section [13 (link)].

Titanium (IV) isopropoxide (TTIP, 97%), chitosan (CS, low molecular weight), potassium ferricyanide (III) [K₃Fe(CN)₆], potassium hexacyanoferrate trihydrate [K₄Fe(CN)₆·3H₂O], N-(3-(dimethylamino)propyl)-N’-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (NHS), and bovine serum albumin (BSA) were acquired from Sigma-Aldrich, Burlington, MA, USA. Potassium chloride (KCl), sodium phosphate monobasic dehydrate (NaH₂PO₄·2H₂O), sodium phosphate dibasic (Na₂HPO₄), and sodium hydroxide (NaOH) were bought from Merck, India. Glacial acetic acid (CH₃COOH), sulfuric acid (H₂SO₄), orthophosphoric acid (H₃PO₄), and hydrochloric acid (HCl) were procured from RANKEM. SARS-CoV-2 nucleocapsid protein (ab273530), and anti-nucleocapsid SARS-CoV-2 immunoglobulin (Ig) G (ab273167) were procured from Abcam, UK. The rest of the solvents and chemicals were of analytical grade and used without further refining. All the solutions were prepared by ultrapure milli-Q water (18.3 MΩ) generated from a Millipore instrument.