Immunohistochemistry for Identifying the Locus Coeruleus Region in Mouse Brain Sections

Samenvatting

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

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.
3. Cut the animal with surgical scissors, snipping the abdominal skin and cutting through the skin in the thorax region. Pin the skin down using T-pins. Then, break the peritoneal membrane up to the thorax. Expose the heart by cracking the thoracic cavity and cutting the diaphragm.
4. Cut the right atrium to allow the blood to flow out of the animal. Insert a 10 mL syringe with a 25-gauge needle into the left ventricle and perfuse with 10 mL of phosphate-buffered saline (PBS).
   NOTE: This allows the solution to flow through systemic circulation and exit through the right atrium.
5. Remove the 10 mL syringe and insert a 25-gauge needle attached to the 60 mL syringe. Perfuse through the left ventricle with 50 mL of ice-cold 4% paraformaldehyde (PFA).
   1. Prepare ice cold 4% PFA solution by diluting 10% PFA solution in H₂O and chilling the final 4% PFA solution at 4 °C.
6. Isolate the head of the mouse and remove the brain from the skull.
   1. Cut the skin from the neck, and then cut towards the eyes to expose the skull. Crack the skull from the neck to the nose, and then from one eyeball to the other. Peel the skull out and excise the whole brain.
7. Incubate the brain in 4% PFA for 24 h at 4 °C.
8. Transfer the brain with forceps into a 50 mL conical tube filled with 25 mL of 30% sucrose solution. Keep it at 4 °C for 48-72 hours until the brain sinks to the bottom of the tube.
9. Cut the brain with an adult mouse brain slicer matrix coronal through the midbrain (~3 mm posterior of bregma). Keep the brain section containing the brainstem.
   NOTE: This will result in two brain sections – one containing most of cortex (anterior of the cut) and one containing the brainstem/cerebellum (posterior of the cut). Use the brainstem section for the following steps.
10. Embed the brainstem section with the cut surface placed on the bottom of an embedding mold, surrounded by optimal cutting temperature compound (OCT); move the embedded brain to a -80 °C freezer and freeze for at least 12 h – until further use.
11. Cutting at the cryostat: place the embedding mold containing the brain in OCT into the cryostat; incubate it in the cryostat for several hours to adjust the temperature of the brain block to that of the cryostat.
12. Peel away the embedding mold to expose the OCT block containing the brain.
13. Use razor blades to remove excess OCT from the surface of the block without touching the brain.
14. Mount the OCT block on the chuck of the cryostat, exposing the cut surface of the brain toward the front.
15. Adjust the cut surface of the brain so that it is oriented in parallel to the razorblades of the cryostat.
16. Trim the brain beginning at the medulla, cutting 100 µm sections rostrally.
17. Trim rostrally until the cerebellum and brainstem are cut as one continuous slice. Begin collecting slices at 50 µm thickness.
    NOTE: As one trims rostrally from the medulla, the brainstem and cerebellum will be cut as two separate sections. In the rostral sections, the brainstem and cerebellum will eventually merge at the level of the 4th ventricle. Once the lateral edges of the 4th ventricle are well-formed, then the cerebellum and brainstem will come out as one continuous slice.
    1. Collect an OCT-surrounded brain slice with forceps and place it in a well of a 24-well plate filled with PBS (Figure 1a). The locus coeruleus (LC) will be most prominent when the cerebellum and inferior colliculus meet one another at ~-5.52 mm posterior of bregma (Figure 2b).
       NOTE: The most anterior part of LC will disappear once the cerebellum has been fully sectioned and no longer surrounds the inferior colliculus at ~-5.34 mm posterior of bregma (Figure 2c).

2. Immunohistochemistry for Dopamine β-Hydroxylase (DBH) or Tyrosine Hydroxylase (TH) (Figure 1)

1. Day 1
   1. Wash the selected slices three times for 5 min in PBS.
   2. Permeabilize for 24 h in 0.5% phosphate-buffered saline with detergent (PBSD) at 4 °C.
      1. Dilute 125 µL of detergent in 25 mL of PBS.
2. Day 2
   1. Wash slices three times for 5 minutes in 0.5% PBSD.
   2. Add the primary antibody, anti-DBH or anti-TH, for 18 h at a dilution of 1:500 in 0.5% PBSD at 4 °C.
3. Day 3
   1. Wash slices three times for 10 min in 0.5% PBSD.
   2. Add the desired secondary antibody (488 donkey anti-rabbit for green fluorescence) at a dilution of 1:1000 in 0.5% PBSD for 16 h.
   3. Wrap the 24-well plate in aluminum and place at 4 °C.
   4. Wash slices three times for 5 minutes in 0.5% PBSD.
   5. Wash for 5 min in PBS.
   6. Transfer slices with a pencil brush into a water container.
   7. Mount slices floating in the water on charged slides.
   8. Coverslip sections with hard-set mounting media Dopamine beta-hydroxylase
   9. Dry the mounted brain sections for 30 min at room temperature.
   10. Image brain slices at a confocal or fluorescent microscope with settings to detect signals from appropriate secondary antibody fluorophore wavelength.
   11. Adjust the microscope to the focal plane of the brain slice and take a single image at 10X magnification.

Resultaten

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...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Adult mouse brain slicer matrix	Zivic Instruments	BSMAS001-1
Anti-rabbit secondary antibody, Alexa Fluor 488 (source - donkey)	Thermo Fisher Scientific	A-21206
Charged glass slides	Genesee	29-107
Confocal microscope	Zeiss	LSM 800
Cryostat	Microm GmbH	HM 505E
Cryostat cutting blades	Thermo Fisher Scientific	MX35
Scissors Mini, 9.5cm	Antech Diagnostcs	503241
DAPI (4',6-diamidino-2-phenylindole)	Sigma-Aldrich	D9542-10MG
Dopamine β-hydroxylase (DBH) antibody - inhouse production (source - rabbit)	B. Eipper	-
Dopamine β-hydroxylase (DBH) antibody - commercially availabe (source - rabbit)	Cell Signaling	8586
Falcon tubes, 50ml	USA Scientific	339652
Forane (isofluorane)	Baxter	NDC 1019-360-60
Forceps Micro Adson	Antech Diagnostcs	501245
Hardset mounting media	EM sciences	17984-24
Microscope	Pascal	LSM 5
Multi-well plates, 24 wells	Thermo Fisher Scientific	930186
Optimal cutting temperature compound (OCT)	VWR/ tissue tech	102094-106
Paraformaldehyde (PFA)/ formalin 10%	Fisher Scientific	SF98-4
Peel-A-Way disposable embedding molds	Polysciences Inc.	18646A
Pencil brush
Phosphate buffered saline (PBS)	Life Tech	14190250
Razor blades	Amazon	ASIN: B000CMFJZ2
Spatulas	Antech Diagnostcs	14374
T pins	Office Depot	344615
The Mouse Brain in Stereotaxic Coordinates, Paxinos and Franklin, 3rd Edition	Amazon	ISBN: 978-0123694607
Triton-X 100 (to prepare PBSD)	Sigma-Aldrich	T8787
Tween 20	Sigma-Aldrich	P7949-500ml
Tyrosine hydroxylase (TH) antibody (source - rabbit)	EMD Millipore	AB152
Wide-field fluorescent microscope	Zeiss	Axio Zoom.V16