Dec 16, 2024
Immunohistochemistry with Rodent Brain Sections
- Mukesh Kumar1,
- Yumei Wu2,3,4,
- Pietro De Camilli2,3,4,
- Timothy A. Ryan1,2
- 1Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland 20815, USA;
- 3Department of Neuroscience, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06520, USA;
- 4Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA
Protocol Citation: Mukesh Kumar, Yumei Wu, Pietro De Camilli, Timothy A. Ryan 2024. Immunohistochemistry with Rodent Brain Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l292kpv1y/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: November 27, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 114543
Keywords: ASAPCRN
Funders Acknowledgements:
NIH
Grant ID: NS036942
NIH
Grant ID: NS11739
ASAP
Grant ID: ASAP-024404
Abstract
This protocol describes a detailed stepwise procedure of immunohistochemical analysis of rodent brain tissue, enabling detailed visualization of target antigens within regions of the hippocampus.
Guidelines
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
Ketamine hydrochloride/xylazine hydrochloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #K113
Fixative: 4% Formaldehyde + 0.125% Glutaraldehyde in 0.1 M phosphate buffer
Formaldehyde solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #F8775-25ML + Glutaraldehyde 50% EM Grade AqueousElectron Microscopy SciencesCatalog #16320 in 0.1 M phosphate buffer (PB), warmed to 37 °C
Normal goat serum: Normal Goat SerumJackson ImmunoResearch Laboratories, Inc.Catalog #005-000-121
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4503
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787
Primary antibodies: DDHD2 Polyclonal antibodyProteintechCatalog #25203-1-AP , Synapsin1/2 antibodySynaptic SystemsCatalog #106 004
Alexa-conjugated secondary antibodies (Thermofisher Scientific)
ProLong™ Gold Antifade MountantInvitrogen - Thermo FisherCatalog #P36934
Nail polish (clear)
Equipments:
• Dissection tools
• Perfusion setup
• Vibratome
Procedure
Procedure
4h 30m
4h 30m
Prepare the anesthetic cocktail (Ketamine/xylazine) and administer it to the mouse via intraperitoneal injection to achieve deep anesthesia.
Note
Confirm the absence of reflexes before proceeding.
Perform transcardial perfusion with 37 °C warmed fixative (4% formaldehyde + 0.125% glutaraldehyde in 0.1 Molarity (M) PB) until the liver and lungs blanch, indicating successful perfusion.
Carefully dissect the brain and immerse it in the same fixative Overnight at 4 °C .
Wash the fixed brain in 0.1 Molarity (M) PB to remove excess fixative.
Use a vibratome to cut coronal sections at 25 μm thickness. Collect sections in 0.1 Molarity (M) PB.
Prepare the blocking solution containing 2% normal goat serum, 2% BSA, 0.1 M PB, and 0.4% Triton X-100 and incubate brain sections in the blocking solution for 01:00:00 at Room temperature with gentle agitation.
1h
Dilute the primary antibodies in the blocking buffer and incubate the sections with the primary antibody solution Overnight at 4 °C .
1h
Wash the sections 3 times in 0.1 M PB, 10 minutes per wash, at room temperature.
Wash the sections in 0.1 Molarity (M) PB for 00:10:00 at Room temperature (1/3).
10m
Wash the sections in 0.1 Molarity (M) PB for 00:10:00 at Room temperature (2/3).
10m
Wash the sections in 0.1 Molarity (M) PB for 00:10:00 at Room temperature (3/3).
10m
Incubate the sections with Alexa-conjugated secondary antibodies prepared in the blocking buffer for 02:00:00 at Room temperature with gentle agitation in the dark to protect fluorescent dyes from photobleaching.
2h
Rinse the sections briefly in 0.1 Molarity (M) PB and mount on microscope slides.
Add ProLong Gold Antifade Reagent to each section and cover with a coverslip.
Seal the edges of the coverslip with nail polish to prevent drying.
Images were acquired with the Yokogawa spinning disk field scanning confocal system with microlensing (63x, oil immersion objective lens, CSU-W1 SoRa, Nikon) controlled by NIS elements (Nikon) software (RRID:SCR_014329).
Note
• Ensure all steps are performed with care to avoid tissue damage.
• Optimize primary and secondary antibody dilutions for specific targets.
• Protect fluorescently labeled sections from light exposure during and after staining.
• Perform controls to verify antibody specificity and avoid background staining.