Apr 24, 2026
Version 2
Immunofluorescence and confocal imaging in brain sections and image analysis V.2
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Chuyu Chen, Loukia Parisiadou 2026. Immunofluorescence and confocal imaging in brain sections and image analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmm6q6v3p/v2Version created by Chuyu Chen
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: April 24, 2026
Last Modified: April 24, 2026
Protocol Integer ID: 315708
Keywords: ASAPCRN, lrrk2 expression in snc dopamine neuron, vulnerable ventral dopamine neuron, distinct dopamine neuronal subtype, dopamine neuron, snc dopamine neuron, distinct dopamine, resilient dorsal neuron, fibers in the dorsolateral striatum, mouse brain section, confocal imaging in brain section, neuronal subtype, aldh1a1 marker, neuron, image analysis single cell profiling study, important insights into cell, dorsolateral striatum, preclinical mouse model, brain section, single cell profiling study, confocal imaging, lrrk2, postmortem pd tissue, immunofluorescence, calbindin1, imaging, dopamine, mouse brain
Funders Acknowledgements:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: Grant ID: ASAP-020600
Abstract
Single cell profiling studies and research on both postmortem PD tissues and preclinical mouse models
demonstrated that the Sox6/Aldh1a1 markers molecularly define the vulnerable ventral dopamine neurons, while Calbindin1 (Calb1) marks the resilient dorsal neurons. These distinct dopamine neuronal subtypes not only differ in their molecular profiling but also exhibit unique projection patterns and functional responses. Therefore, studying these neurons in a subtype-specific manner can yield important insights into cell-intrinsic mechanisms of vulnerability in PD. We first addressed whether LRRK2 is expressed in dopamine neurons, as this is a prerequisite for any cell-autonomous effects. We performed immunolabeling on mouse brain sections and confirmed LRRK2 expression in SNc dopamine neurons. We also employed confocal imaging and 3D reconstructions to assess Th+ fibers in the dorsolateral striatum.
Safety warnings
These protocols need prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee
Immunofluorescence
Perfuse mice (5ml/min) with 50 ml of PBS, followed by 50 ml of 4% paraformaldehyde in PBS.
post-fix with 4% paraformaldehyde in PBS, overnight at 4°C
Change buffer with 30% sucrose in PBS, 48 hours
Cryosection (30 μm thickness). The slices were collected in PBS containing 0.1% sodium azide and stored at 4°C for immunohistochemistry.
Incubate brain sections in 5% goat serum with 0.2% Triton X-100 for 2 hours RT.
Antigen retrieval: incubate the sections in antigen unmasking solution (Vector, H-3300, 48ul in 5ml water) at 100°C for 30 minutes
cooling the sections to room temperature, 30 minutes RT
wash with PBS x 3, 5 mins, RT
Incubate section overnight at 4°C in 5% goat serum with 0.2% Triton X-100
incubate with primary antibodies in 5% goat serum with 0.2% Triton X-100, 48-72 hrs at at 4°C
Lrrk2 signal in SNc sections: anti-Th (1:1000, SYSY), anti-Aldh1a1 (1:300, Abcam), anti-LRRK2/Dardarin clone N241A/34 (1:200, NeuroMab), anti-GFP (1:1000, invitrogen).
Striatal Th expressing fibers :: anti-Th (1:1000, SYSY), anti-Aldh1a1 (1:300, Abcam), and anti-Vmat2 (1:200, Immunostar).
washing with PBS x3, 5 mins, RT
incubated with secondary antibodies—Alexa Fluor‱ 488, Alexa Fluor‱ 568, and Alexa Fluor‱ 647 (1:300, invitrogen) in 5% goat serum with 0.2% Triton X-100, 3hrs, RT
wash with PBS 5 mins x3, RT
mount the sections using ProLong‱ Diamond Antifade Mountant (invitrogen)
Confocal imaging
Confocal images are obtained using the Nikon A1R microscope at a resolution of 1,024 x 1,024 pixels, maintaining constant laser power for each channel across genotypes.
20x objective for Lrrk2 expression, Th area, intensity and APEX2 expression,
100x objective for Th
Confocal images are obtained with Nikon CSU-W1 SoRa with a 60x objective at 0.1 mm intervals at 1,024 x 1,024 pixel resolution for volume measurements of Th fibers
Image analysis: The protein signals for Th, Aldh1a1, and Lrrk2 were measured using Imaris 10.1 software (Bitplane, Concord, USA).
Lrrk2 signal in SNc sections
The surface rendering function are used to segment Th/Aldh1a1 cells
Enable background subtraction; the diameter for the largest sphere is set to 10 μm and automatically thresholded, with a smoothing surface set to 3 μm
Mean intensity of Aldh1a1 protein within the Th surface above 1.5 times the average Th protein channel mean intensity is set to be considered positive
The Lrrk2 intensity within Th-positive cells is measured automatically by the software
Striatal Th expressing fibers analysis:
Volume measurements of Th fibers
Surface rendering function is used to segment the Th fiber.
Enable background subtraction; the diameter of the largest sphere is set at 0.805 μm and the threshold set at 2000.
filter segments with “Number of voxels Img=1” set above 50. Th volume are measured automatically by software.
Intensity and area of Th expressing axons.
Performe rolling ball background subtraction with Nikon Elements software.
For the high magnification analysis
Use surface rendering function segment TH, Aldh1a1, and Vmat2 proteins.
Disable background subtraction; automated thresholds are applied with manual adjustments made as needed.
Filter segments with “Number of voxels Img=1” set above 50.
Apply “overlapped area ratio to surface surface=TH-aldh1a1” above 0.4 for the Aldh1a1 positive Th axon,
Filter Vmat2 within Th axonal terminal with “overlapped area ratio to surface surface= Th-VMAT2” above 0.4.
Area and intensity are measured automatically by software.
For the low magnification analysis
Use the surface rendering function segment TH.
Disable background subtraction; automated thresholds are applied with manual adjustments made as needed.
Filter segments with “Number of voxels Img=1” set above 50. Th area and intensity are measured automatically by software.