Oct 28, 2024
Mouse brain immunohistochemistry - colorimetric analysis
- Ilaria Morella1,
- Riccardo Brambilla1
- 1Università di Pavia
Protocol Citation: Ilaria Morella, Riccardo Brambilla 2024. Mouse brain immunohistochemistry - colorimetric analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1md2lmk/v1
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: July 17, 2024
Last Modified: October 28, 2024
Protocol Integer ID: 104414
Keywords: ASAPCRN
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Abstract
This protocol details the mouse brain immunohistochemistry by colorimetric analysis.
Materials
• Euthatal
• PBS
• PFA 4% (v/v) in PBS
• Sucrose 30% (w/v) in PBS
• Quenching solution (3% H2O2, 10% Methanol in TBS)
• Blocking solution (5% normal goat serum, TBS-Triton 0.1%)
• Vector Elite ABC KitContributed by usersCatalog #PK-4000
• 3,3′-diaminobenzidine (DAB, Sigma)
• Primary antibody
Before start
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Procedure:
Procedure:
1d
1d
Anesthetise the mice with Euthatal and perfuse with PBS and PFA 4%.
Fix the brains Overnight in 4% PFA at 4 °C , wash and left in 30% sucrose for 24:00:00 at 4 °C .
1d
Slice the brains into 35 μm-thick slices using a freezing microtome and store at -20 °C until processing for immunohistochemistry.
Rinse the free-floating sections (3X) with TBS for 10 min.
Rinse the free-floating sections with TBS for 00:10:00 (1/3).
10m
Rinse the free-floating sections with TBS for 00:10:00 (2/3).
10m
Rinse the free-floating sections with TBS for 00:10:00 (3/3).
10m
Incubate with quenching solution (3% H2O2, 10% Methanol in TBS) for 00:15:00 .
15m
Subsequently rinse the sections (3X) in TBS for 10 min.
Subsequently rinse the sections in TBS for 00:10:00 (1/3).
10m
Subsequently rinse the sections in TBS for 00:10:00 (2/3).
10m
Subsequently rinse the sections in TBS for 00:10:00 (3/3).
10m
Incubate with blocking solution (5% normal goat serum, TBS-Triton 0.1%) for 01:00:00 at Room temperature .
1h
Perform the incubation with primary antibodies Overnight at 4 °C .
1h
The following day, rinse the sections (3X) with TBS Triton 0.1% for 10 min.
The following day, rinse the sections with TBS Triton 0.1% for 00:10:00 (1/3).
10m
The following day, rinse the sections with TBS Triton 0.1% for 00:10:00 (2/3).
10m
The following day, rinse the sections with TBS Triton 0.1% for 00:10:00 (3/3).
10m
Incubate with the secondary antibodies for 02:00:00 at Room temperature .
2h
Subsequently rinse the sections (3X) in TBS-Triton for 0.1% for 10 minutes.
Subsequently rinse the sections in TBS-Triton for 0.1% for 00:10:00 (1/3).
10m
Subsequently rinse the sections in TBS-Triton for 0.1% for 00:10:00 (2/3).
10m
Subsequently rinse the sections in TBS-Triton for 0.1% for 00:10:00 (3/3).
10m
Incubate for 02:00:00 at Room temperature with avidin-peroxidase complex (ABC kit, PK4000, Vector).
2h
Apply the 3,3′-diaminobenzidine (DAB, Sigma) to the slices to visualise Iba1 and DARPP-32 positive cells.
Obtain the images using a bright-field microscope (Macro/Micro Imaging System, Leica) under a 40X objective and analysed using Fiji.