Method Article

Immunohistochemistry for Identifying the Locus Coeruleus Region in Mouse Brain Sections

July 8th, 2025

In This Article

Abstract

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Source: Schmidt, K. et al., Localization of the Locus Coeruleus in the Mouse Brain. J. Vis. Exp. (2019)

This protocol outlines the use of immunohistochemistry to identify locus coeruleus regions in mouse brain sections. By targeting dopamine β-hydroxylase with green fluorophore-labeled antibodies, norepinephrine-producing neurons are visualized as green fluorescence under a microscope.

Protocol

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Brain Slicing

  1. To immobilize, and anesthetize mice by the application of 3% isoflurane.
    1. Soak a cotton ball with drops of isoflurane and place it in a 15 mL microcentrifuge tube. Place the animal's nose into the tube and allow it to inhale the isoflurane. Check for the depth of anesthesia by the lack of response to toe-pinch.
  2. Place the animal on its back and immobilize it by pinning its extremities down with a T pin while having access to its abdomen.

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Results

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Cell assay results, fluorescence microscopy images, DBH and TH labeling in brain tissue, diagram.
Figure 1: Detection of LC by immunostaining brain slices for DBH or TH. (a) One mouse brain was sectioned into 50 μm slices around the brain stem and collected in two 24-well dishes. Every 5th to 8th collected brain slice was mounted on film covered c.......

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Adult mouse brain slicer matrixZivic InstrumentsBSMAS001-1
Anti-rabbit secondary antibody, Alexa Fluor 488 (source - donkey)Thermo Fisher ScientificA-21206
Charged glass slidesGenesee29-107
Confocal microscopeZeissLSM 800
CryostatMicrom GmbHHM 505E
Cryostat cutting bladesThermo Fisher ScientificMX35
Scissors Mini, 9.5cmAntech Diagnostcs503241
DAPI (4',6-diamidino-2-phenylindole)Sigma-AldrichD9542-10MG
Dopamine β-hydroxylase (DBH) antibody - inhouse production (source - rabbit)B. Eipper-
Dopamine β-hydroxylase (DBH) antibody - commercially availabe (source - rabbit)Cell Signaling8586
Falcon tubes, 50mlUSA Scientific339652
Forane (isofluorane)BaxterNDC 1019-360-60
Forceps Micro AdsonAntech Diagnostcs501245
Hardset mounting mediaEM sciences17984-24
MicroscopePascalLSM 5
Multi-well plates, 24 wellsThermo Fisher Scientific930186
Optimal cutting temperature compound (OCT)VWR/ tissue tech102094-106
Paraformaldehyde (PFA)/ formalin 10%Fisher ScientificSF98-4
Peel-A-Way disposable embedding moldsPolysciences Inc.18646A
Pencil brush
Phosphate buffered saline (PBS)Life Tech14190250
Razor bladesAmazonASIN: B000CMFJZ2
SpatulasAntech Diagnostcs14374
T pinsOffice Depot344615
The Mouse Brain in Stereotaxic Coordinates, Paxinos and Franklin, 3rd EditionAmazonISBN: 978-0123694607
Triton-X 100 (to prepare PBSD)Sigma-AldrichT8787
Tween 20Sigma-AldrichP7949-500ml
Tyrosine hydroxylase (TH) antibody (source - rabbit)EMD MilliporeAB152
Wide-field fluorescent microscopeZeissAxio Zoom.V16

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Tags

Dopamine Beta HydroxylaseNorepinephrine NeuronsFluorescence MicroscopyPrimary AntibodiesSecondary AntibodiesConfocal MicroscopyBrainstem Sections

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