Donkey anti goat igg

Myelin was detected using luxol fast blue stain (LFB; Sigma) followed by cresyl violet counterstain (Sigma) to identify neuronal tissue structure. The guideline for scoring is shown in S1 Fig. Astrocytes were detected using anti-glial fibrillary acid protein (GFAP, 1:1000 DAKO, Glostrup, Denmark) overnight at 4°C followed by donkey anti-rabbit IgG at 1:500 (Jackson Immunochemicals, PA, USA) for 3 hours. Slides were then stained with an ABC staining kit (Vector Labs, CA, USA) followed by Vector VIP substrate (Vector) according to the manufacturer’s instructions. Microglia were detected using ionized calcium binding adaptor molecule 1 (Iba1, 1:250 Abcam, Cambridge, UK) and myelin was detected using myelin basic protein (MBP, 1:500 Millipore, Burlington, MA, USA). Both antibodies were incubated on sections overnight at 4°C followed by donkey anti-goat IgG and donkey anti-rat IgG at 1:500 (Sigma) for 3 hours. Sections were then stained with extravidin peroxidase (1:500, Sigma) for 1 hour and developed with diaminobenzidine/nickel sulfate (DAB/Ni, Sigma) for up to 20 minutes. Sections of corpus callosum were assessed using a photomicroscope with an Olympus DP72 camera (Tokyo, Japan) and CellSens software (version 1.16; Olympus, Tokyo, Japan) at 20 x magnification.

After treatment, cells were fixed in 4% paraformaldehyde in PBS. Microglia were then stained with rabbit anti-Iba1 (1:1000, Wako, Japan), mouse anti-Cy5 (1:2000, GE Healthcare, UK) and rat anti-CD68 (1:1000, Abcam, UK), all diluted in 1% bovine serum plus 1% Triton in 0.1 N PB. For Tau membrane binding assays, antibodies were diluted in the same buffer without Triton. Cells were then incubated with a donkey anti-goat IgG conjugated to Alexa Fluor 488, donkey anti-rat IgG conjugated to Alexa Fluor 555, donkey anti-mouse IgG conjugated to Alexa Fluor 647 (1:1000), and DAPI (Merck, USA) at 1:10,000 dilution.

Acid citric dextrose (ACD) tubes were from BD Vacutainer. Ficoll-Paque was purchased from GE Healthcare. Azidohomoalanine (AHA), bicinchonic acid assay kit, Click-it protein reaction kit, biotin alkyne probe, dithiothreitol (DTT), Triton-X100m, C18 tips, goat polyclonal anti-biotin and goat anti-rabbit Alexa FluorVR 680 IgG antibodies were from ThermoFisher. RPMI 1640 Met^-, P8340 protease inhibitor cocktail, magnetics streptavidin beads, ammonium bicarbonate (ABC), iodoacetamide (IAA), formic acid (FA), mouse monoclonal anti-actin (clone AC-15), Donkey anti-goat IgG, and goat anti-rabbit IgG HRP-conjugated antibodies were bought from MilliporeSigma. Trypsin/lys-C and the enhanced chemiluminescence kit (ECL) were from Promega and PerkinElmer respectively. Rabbit monoclonal anti-ILK (EP1593Y), rabbit monoclonal anti-ANXA2 (ERP13052B) and mouse monoclonal anti-FERMT3 (3D6) were from Abcam. Mouse monoclonal anti-ATP2A3 (PL/IM430) was from Santa Cruz Biotechnology. Recombinant chicken anti-VCL antibody was from Immune Biosolutions. Goat anti-mouse IgG HRP-conjugated was from Jackson IR. Goat anti-mouse IRDyeVR 800CW IgG was from LI-COR Biosciences.