Polyclonal goat

GFAP and CHI3L1 expression was studied in the brain of 7 CSVD and 5 control cases without neurological disorders (Table 2). The brains of all autopsied subjects were fixed in a 4% formaldehyde solution and underwent a routine neuropathological examination, as described previously [34 (link),35 (link)]. A coronal mid-hemispheric block (approx. 1 cm thick) was embedded in polyethylene glycol (PEG 1000, Merck, Carl Roth Ltd., Karlsruhe, Germany). Multiple 100-µm-thick consecutive sections were obtained from each block with the aid of a sliding microtome (Jung, Heidelberg, Germany). Immunohistochemistry (IHC) was performed with primary antibodies against GFAP (1:1000, monoclonal mouse, Abcam, Cambridge, UK) or CHI3L1 (1:750, polyclonal goat, R&D Systems, Exton, PA, USA) and a secondary biotinylated antibody (1:200; 2 h, room temperature, Vector Laboratories, Burlingame, CA, USA). The immunohistochemical reaction was visualised with an avidin–biotin–peroxidase complex (ABC Vectastain Kit, Vector Laboratories, Burlingame, CA, USA) and the chromogen 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma Taufkirchen, Germany), which was intensified using Cobalt(II)Chloride (32 nM) for the labelling of CHI3L1. Omission of the primary antibody resulted in a lack of staining.