Immunohistochemistry of rTg4510 mouse brain
Itika Saha, F. Ulrich Hartl, Mark S. Hipp, Miguel Da Silva Padilha, Irina Dudanova
Abstract
This protocol describes immunohistochemical staining of the Tau transgenic mice rTg4510 (Santacruz et al, 2005) brain section for Tau aggregates phosphorylated at Ser202/Thr205 and the AAA+ ATPase Valosin-containing protein (VCP). Experiments involving animal models must be performed in accordance with relevant institutional guidelines and regulations.
Steps
Deeply anesthetize mice with 1.6% Ketamine/0.08% Xylazine and transcardially perfuse with PBS followed by 4% paraformaldehyde (PFA)(Santa Cruz) in PBS.
Dissect brains out of the skull and post-fix in 4% PFA in PBS overnight.
Embed fixed tissue in agarose and section into 40 μm thick sections using a vibratome (VT1000S, Leica).
Permeabilize sections with 0.5% Triton X-100 and wash with PBS.
Incubate sections in blocking solution consisting of 0.2% BSA (w/v), 5% donkey serum (v/v) (abcam), 0.2% lysine (w/v) (Sigma-Aldrich), 0.2% glycine (w/v) (Sigma-Aldrich) in PBS for 30 min at room temperature (RT).
Incubate sections with primary antibodies (anti-phospho-Tau AT-8 (Thermo, Cat# MN1020, 1:300); anti-VCP (Novus Biologicals, Cat# NB 100-1558, 1:500) diluted in 0.3% Triton X-100 (v/v), 2% BSA (w/v) in PBS overnight at 4°C.
Wash sections in PBS and incubate with secondary antibodies and Neurotrace 640/660 (ThermoFisher, Cat# N21483, 1:500) diluted in 0.3% Triton X-100, 3% donkey serum (v/v) for 2 h at RT.
Stain nuclei with 0.5 µg/ml DAPI.
Mount sections on Menzer glass slides using Prolong Glass fluorescence (Invitrogen) mounting medium.
