Sep 06, 2022

Public workspaceImmunohistochemistry of rTg4510 mouse brain

DOI
dx.doi.org/10.17504/protocols.io.x54v9den4g3e/v1
  • Miguel Da Silva Padilha1,
  • Irina Dudanova1,
  • Itika Saha2,
  • F. Ulrich Hartl2,
  • Mark S. Hipp3,4
  • 1Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, 82152 Martinsried, Germany;
  • 2Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
  • 3Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan, 1, 9713 AV Groningen, The Netherlands;
  • 4School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, 26129 Oldenburg, Germany
Felix Kraus
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DOI: dx.doi.org/10.17504/protocols.io.x54v9den4g3e/v1
External link: https://www.biorxiv.org/content/10.1101/2022.02.18.481043v1.full
Protocol Citation: Miguel Da Silva Padilha, Irina Dudanova, Itika Saha, F. Ulrich Hartl, Mark S. Hipp 2022. Immunohistochemistry of rTg4510 mouse brain. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9den4g3e/v1
Manuscript citation:
Itika Saha, Patricia Yuste-Checa, Miguel Da Silva Padilha, Qiang Guo, Roman Körner, Hauke Holthusen, Victoria A. Trinkaus, Irina Dudanova, Rubén Fernández-Busnadiego, Wolfgang Baumeister, David W. Sanders, Saurabh Gautam, Marc I. Diamond, F. Ulrich Hartl, Mark S. Hipp bioRxiv 2022.02.18.481043; doi: https://doi.org/10.1101/2022.02.18.481043
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 05, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69594
Keywords: ASAPCRN
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.
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.
12h
Embed fixed tissue in agarose and section into 40 μm thick sections using a vibratome (VT1000S, Leica).
Note
NOTE: The sections can be stored in PBS 0.05% sodium azide at 4°C.

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).
30m
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.
12h
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.
2h
Stain nuclei with 0.5 µg/ml DAPI.
Mount sections on Menzer glass slides using Prolong Glass fluorescence (Invitrogen) mounting medium.