Feb 11, 2026
4.5x Expansion of Mouse Brain tissue sections
- Finn Funk1,
- Suzanne Pfeffer2
- 1Stanford University School of Medicine;
- 2Stanford University School of Medicine and Aligning Science Across Parkinson's
- Finn Funk: Current Address: Freie Universität Berlin;
Protocol Citation: Finn Funk, Suzanne Pfeffer 2026. 4.5x Expansion of Mouse Brain tissue sections . protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp15w1gzp/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: February 04, 2026
Last Modified: February 11, 2026
Protocol Integer ID: 242585
Keywords: Expansion microscopy, primary cilia, brain IHC, mouse brain tissue section, mouse brain tissue, positive cilia of striatal cholinergic interneuron, mouse brain slice, striatal cholinergic interneuron, embedding brain slice, brain slices in swellable acrylamide gel, subcellular targets in the brain, enlarged arl13b, subcellular target, brain, traditional microscopy, swellable acrylamide gel, enhanced resolution
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP 000463
Abstract
This technique expands mouse brain tissue 4.5-fold in all spatial dimensions by embedding brain slices in swellable acrylamide gels. This allows enhanced resolution compared to traditional microscopy to precisely investigate subcellular targets in the brain. Here we adapted previously developed expansion protocols to reliably image enlarged Arl13b-GFP-positive cilia of striatal cholinergic interneurons in Arl13b-GFP knock-in mouse brain slices.
Materials
Troubleshooting
Safety warnings
[NOTE: because this chamber will be incubated at 37°C for prolonged times, it should be kept as clean as possible using 70% ethanol spray.]
[NOTE: use care when washing; in between washes, use a pipet tip to carefully remove buffer].
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Protocol
Prepare all stock solutions in ddH₂O and then prepare 10ml monomer solution.
Make 500 µl aliquots of monomer solution and store at − 20°C. Thaw on ice and vortex before use.
Prepare 10% (v/v) TEMED in ddH2O. Store at 4°C.
Prepare 10% APS (w/v) in ddH2O. Can be stored at 4°C but loses activity with time.
Prepare 1% 4-hydroxy-TEMPO (w/v) in ddH2O. Solution can be aliquoted and frozen at -20°C.
Prepare homogenization buffer [store at room temperature to avoid precipitation].
Prepare gelation chamber and glass slide chamber
Recycle a 1ml pipet tip box; cover holes with Parafilm and use tape to secure it to the box; leave some holes open. Fill the bottom of this chamber with sterile water to create a humid environment. [NOTE: because this chamber will be incubated at 37°C for prolonged times, it should be kept as clean as possible using 70% ethanol spray.]
Figure 1: Gelation chamber made out of a 1 mL tip box.
Glass slide chambers are created by gluing 22x22mm micro coverslips to each side of the slide using Super glue (see Figure 2).
Figure 2: Schematic of brain tissue on microscope cover slide chamber prior to gelation.
Mouse brain preparation
Mouse brains are fixed by transcardial perfusion using 4% paraformaldehyde (PFA) in PBS.
Next, whole brain tissue is surgically extracted from the skull, post-fixed in 4% PFA for 24 hr and then immersed in 30% (w/v) sucrose in PBS until the tissue settled to the bottom of the tube (~48 hr).
Prior to cryosectioning, brains are embedded in cube-shaped plastic blocks with Tissue-Tek® OCT Compound and stored at −80°C.
OCT blocks are allowed to reach −20°C for ease of sectioning and 30µm slices are cut and stored at 4°C in PBS.
Gel embedding, staining and imaging
The gelation chamber is brought to a cold room (4°C) and cooled. Monomer solution is thawed on ice (~40 min).
Brain tissue sections are transferred to glass coverslip chambers using a paintbrush and washed 3 times with PBS. [NOTE: use care when washing; in between washes, use a pipet tip to carefully remove buffer]. The slice is washed one more time with PBS and left to air dry for 3 min at 4°C (in cold room).
Add 0.56 µl methacrolein (95% stock) and 0.56 µl, 1% 4-hydroxy-TEMPO (0.001% final) are added to 500µL monomer solution. Next, 27.7µL, 10% TEMED in ddH2O and 27.7µL, 10% APS in ddH2O are added and the mixture is quickly vortexed. Cover the tissue completely by carefully pipetting the mix onto the brain section. Another slide is used to cover the sample and gelation solution, while avoiding introducing bubbles to the gel.
The gel is left to polymerize for 30min at 4°C in the humidified gelation chamber in the cold room (Figure 1). Next, the chamber is transferred to a 37°C incubator overnight. The next day, the top glass slide is carefully removed and the gel containing the brain slice is trimmed to remove excess gel.
The glass slide with gel attached is next transferred to a 10cm dish containing homogenization buffer. The gel will slowly detach from the coverslip and can then be transferred to a 2 mL Eppendorf tube.
The tube is then filled with homogenization buffer to cover the gel completely and transferred to a Thermo-Shaker for 1.5h at 85°C.
The gel is next transferred to a 10cm dish and washed 3X with 10mL PBS-T (1X PBS with 1% Tween-20) for 10 min each, with gentle shaking in an orbital shaker. The buffer is then aspirated and the gel transferred to a 2 mL tube using clean gloves.
Primary antibody is diluted in 1mL, 2% BSA (1:1000 for anti-GFP antibody but other antigens may require 1:200) in PBS and added to the tube. Tubes are incubated with rotation for 3h or overnight at RT.
Gels are removed from the tubes and washed again 3X in PBS in a 10 cm dish with shaking.
Secondary antibody (1:2000) and DAPI (1:300) staining is carried out for 3h or overnight at RT in 2mL tubes.
Gels are washed 3X for 10 min each with PBS in a 10cm dish.
To expand the gel, wash it at least 3X in 15 mL ddH2O for 30 min each on an orbital shaker.
The expanded gel is carefully cut into pieces that fit into 6-well glass bottom plates. The gels can then be imaged using a Zeiss LSM 900 with a 20x air objective/63x Oil objective.
Figure 3: Expansion microscopy images of a mouse brain slice expressing Arl13b-GFP (green), stained for choline acetyltransferase (ChAT) (red) and DAPI (cyan). At left are 2 examples from the striatum. The enlarged image below right shows cilia labeled with anti-Arl13b where distinct ciliary
membranes can be detected. Above right shows a low magnification image of the brain section. NOTE THAT THESE MICE HAVE VERY STRANGE LOOKING CILIA--extremely elongated.
Protocol references
The monomer solution is adapted from Gambarotto et.al. (2019), with the implementation of the molecular anchoring strategy introduced by Kimas et.al. (2023).
Gambarotto, D., Zwettler, F.U., Le Guennec, M. et al. Imaging cellular ultrastructures using expansion microscopy (U-ExM). Nat Methods 16, 71–74 (2019). https://doi.org/10.1038/s41592-018-0238-1
Klimas, A., Gallagher, B.R., Wijesekara, P. et al. Magnify is a universal molecular anchoring strategy for expansion microscopy. Nat Biotechnol 41, 858–869 (2023). https://doi.org/10.1038/s41587-022-01546-1