Apr 02, 2026
Mouse brain immunofluorescence
- Yumei Wu1,2,
- Pietro De Camilli1,2
- 1Departments of Neuroscience and of Cell Biology, Program in Cellular Neuroscience, Neurodegeneration, and Repair, Howard Hughes Medical Institute, Yale School of Medicine, New Haven, CT 06510.;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network.
Protocol Citation: Yumei Wu, Pietro De Camilli 2026. Mouse brain immunofluorescence. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn515zg5d/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: April 02, 2026
Last Modified: April 02, 2026
Protocol Integer ID: 314451
Keywords: ASAPCRN
Abstract
This protocol details the procedure of our mouse brain immunofluorescence for detecting DAergic axonal dystrophies in Synj^^RQ KI mice.
Troubleshooting
General preparation
Mouse is anesthetized with a Ketamine/Xylazine anesthetic cocktail (16 mg/ml Ketamine and 0.8 mg/ml Xylazine) injection.
Perfused transcardially with 37 °C pre-warmed 4% PFA and 0.05% Glutaraldehyde in 0.1M Phosphate buffer (PB) (0.1M Na2HPO4+0.1M NaH2PO4, PH7.4) at 5-6 ml/min with a pump.
Dissect the brain out and keep in the same fixative overnight at 4 °C.
Wash 3x20min in 0.1M PB buffer.
SectionCoronal or sagittal sections of 25-30 µm thickness is with a vibratome.
The floating sections are then blocked in 2% BSA, 2% donkey/goat serum, 0.4% Triton X-100, 0.05% Tween20, 0.01% NaN3, and 50mM NH4Cl in 0.1M PB buffer for 2 h at room temperature.
Incubated the sections with primary antibodies (diluted in the same buffer) and keep overnight at 4 °C.
Wash 10minx3 in 01M PB,
Incubated with Alexa-conjugated secondary antibodies for 2 h at room temperature.
Mount the sections onto glass slides with ProLong^^TM^^Gold antifade reagent with or without DAPI (Invitrogen, P36934) and sealed with nail polish.
Acquire fluorescence images either with an Olympus slice view VS200 slide scanner equipped with a Hamamatsu Orca-Fusion camera and a 20x Olympus UPlanXApo objective or a Nikon Ti2-E inverted microscope (Yokogawa CSU-W1 SoRa, Nikon) equipped with a 60x SR Plan Apo IR oil-immersion objective.