Anti c3d a0063

PLA™ was performed with Duolink^® In Situ Orange Starter Kit Mouse/Rabbit (DUO92102-1KT) following manufacturer’s protocol. The primary antibodies couples used were: anti-C3d (A0063, DAKO) and anti-GRP94 (ab210960, Abcam, Cambridge, UK), anti-C3d (A0063) and anti-HSP105 (sc-74550, SantaCruz Biotechnology, Dallas, Texas, USA), anti-GRP94 (ab210960) and anti-TLR4 (56580, NOVUS Biological, Centennial, Colorado, USA) and anti-cathepsin-L (PA5-88413, ThermoFisher Scientific, Waltham, Massachusetts, USA). Microscopy images were taken on an Axio Imager 2 (Carl Zeiss Microscopy GmbH, Jena, Germany). Images were acquired using an AxioCam MRm monochrome CCD camera (Carl Zeiss GmbH) and analyzed using ImageJ^® software. Spotted quantification was performed using Icy^® software.

Slices were deparaffined with absolute Xylene (3 × 5 min) and rehydrated by scaling down from 100% to 50% ethanol, 2 × 10 min for each concentration. After PBS washes (3 × 5 min), antigen retrieval has been performed by soaking slices for 40 min in a warm solution containing (in mM): 10 Na-citrate, 0.05% Tween 20, pH 6.0, 90°C. Subsequently, slices were incubated for 45 min in a blocking solution (3% goat serum and 0.3% Triton X-100 in PBS). Primary antibodies diluted in blocking solution were incubated over-night at 4° [anti-βTubulin III, T2200, Sigma, clone Aa441-450, 1:500; anti-GFAP, MAB360, Millipore, clone GA5; 1:400; anti-Iba1, 019-19741, Wako, 1:400; anti-OPN, 691302, Biolegend, clone A15059B, 1:100; Anti-βAmyloid, 8243s, CELL SIGNALING, clone D54D2, 1:100; anti-Cleaved-Casapase3, 9661s, CELL SIGNALING, clone Asp175, 1:100; anti-PhosphoTau (Thr212, Ser214), MN106, INVITROGEN clone AT100 1:50; anti-PhosphoTau (Ser 202, Thr205), INVITROGEN, clone AT8, 1:40; anti-C3d, A0063, Dako, 1:500; anti-IL-1β, sc-32294, Santa Cruz Biotechnology, clone E7-2-hIL1β, 1:50]. After three washes in PBS, sections were stained for 45 min with a secondary antibody and Hoechst in order to visualize nuclei, then coverslips were mounted with Diamond Antifade Mountant (Molecular Probes) and images acquired with a confocal microscopy.