Goat anti human c1q

To measure C1q levels in the brain, 150 μg of brain lysate in 1×PBS was added to Corning® 96-well black flat bottom polystyrene high bind microplate (Steuben County, NY) and incubated overnight at 4°C. The plates were blocked with 10% normal donkey serum (NDS) for 1 hour after being washed with 1x PBS-0.1% Tween 20 (PBST) three times. Goat anti-human C1q (Complement Technology, Inc., Tyler, TX) at 1:50 in NDS was added to the wells and incubated at RT for 1 hour. After washing, donkey anti-goat Alexa Fluor (AF) 488 (Life Technologies, Grand Island, NY) at 1:500 dilution in NDS was added followed by a 1 hour incubation at RT. The plate was washed and blocked with 10% normal goat serum (NGS) at RT over 1 hour. The wells were washed, and the plate read using a Synergy HT (BioTek, Winooski, VT).

Primary antibodies used for assays: Goat anti-rat C3 IgG (MP Biomedicals, Santa Ana, CA, United States), Chicken anti-C3/C3a (Abcam, Cambridge, MA, United States), Rabbit anti-rat C9 (generously provided by Professor Paul Morgan, Cardiff, United Kingdom), goat anti-human C1q (Complement Technology, Inc., Tyler, TX, United States), mouse anti-NeuN (EMD Millipore, Temecula, CA, United States), chicken anti-NeuN (EMD Millipore), mouse anti-GFAP (Sigma-Aldrich, St. Louis, MO, United States), chicken anti-GFAP (Abcam), goat anti-Iba1 (Abcam), rabbit anti-C3aR (Bioss Inc., Woburn, MA, United States), Mouse anti-C5aR (Hycult Biotech., Plymouth Meeting, PA, United States). Secondary antibodies included goat anti-mouse horseradish peroxidase (HRP) (Sigma-Aldrich), goat anti-chicken IgY HRP (Genway Biotech, San Diego, CA, United States), goat anti-rabbit HRP (Sigma-Aldrich), rabbit anti-goat HRP (Thermo Fisher Scientific, Grand Island, NY, United States), biotinylated goat anti-mouse IgG (H + L), biotinylated goat anti-chicken IgG (H + L) (Vector Laboratories, Burlingame, CA, United States), biotinylated goat anti-rabbit IgG (H + L) (Bioss Inc), donkey anti-goat IgG (H + L) Alexa Fluor (AF) 488/568, goat anti-chicken IgG (H + L) AF 488, goat anti-mouse IgG (H + L) AF 568, and goat anti-rabbit IgG (H + L) AF 405/488 (Life Technologies, Grand Island, NY, United States).