Anti mouse igg gold secondary antibody

Following PBS perfusion, mouse brainstem tissue was dissected and frozen in liquid nitrogen. Sarkosyl extraction was performed as previously described (Delobel et al., 2008 (link)). Briefly, the brainstem tissue was homogenized in A68 buffer (0.5 ml of 800 mM NaCl, 10% sucrose, 10 mM Tris-HCl pH 7.4, 1 mM EGTA) using a Kinetica polytron. Samples were centrifuged at 5000g for 15 min. The collected supernatant was analysed as total tau samples. Following sarkosyl addition to 1% and shaking for 1 h, samples were centrifuged at 80 000g for 30 min. The resulting pellet was resuspended in 50 mM Tris-HCl pH 7.4.
For immunoelectron microscopy, aliquots were placed on carbon-coated 400 mesh grids and allowed to dry partially. Grids were blocked in droplets of 0.1% gelatin (Sigma G7041, Sigma-Aldrich) and stained with HT7 primary antibody (1:50; Pierce). Grids were then washed briefly with blocking buffer, stained with anti-mouse IgG-Gold secondary antibody (Sigma G7652), washed with water, and stained with 2% uranyl acetate. Electron microscopy was performed using a FEI Tecnai Spirit TEM at a magnification of ×21 000 and images recorded using a Gatan Orius SC200B CCD camera (Gatan).

Cells were fixed in 0.1% glutaraldehyde and 4% paraformaldehyde in 0.1 mol l⁻¹ phosphate buffer for 2 h and were then collected and centrifuged at 900g for 5 min. For cryoprotection, cells were incubated with 2.3 mol l⁻¹ sucrose in 0.1 mol l⁻¹ phosphate buffer overnight and then frozen in liquid nitrogen. Thin cryosections (60 nm) were cut using the Leica cryo-microtome. The sections were incubated with primary antibodies (1:20) at 4 °C overnight. The sections were then incubated with a 10 nm anti-mouse IgG gold secondary antibody (Sigma-Aldrich, G7652) for 2 h at room temperature. After washes, the sections were fixed in 1% glutaraldehyde and embedded in 2% methyl cellulose solution containing 0.3% uranyl acetate. The samples were imaged using the Jeol 1200 electron microscope (Mayo Clinic Core Microscopy Facility) operating at 60 to 80 kV.