Mar 28, 2023
Expansion microscopy
- michela.deleidi1,
- María José Pérez J.1,
- Hariam Raji1,
- Pascale Baden1,
- Federico Bertoli1
- 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Protocol Citation: michela.deleidi, María José Pérez J., Hariam Raji, Pascale Baden, Federico Bertoli 2023. Expansion microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg39n5qg25/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: March 17, 2023
Last Modified: March 28, 2023
Protocol Integer ID: 78968
Keywords: Expansion microscopy
Abstract
Expansion microscopy is a technique to visualize biological structures with higher spatial resolution than traditional microscopy methods.
Attachments
676- 1424.docx
13KB
Materials
Materials
Stock X solution
| A | B | |
| Sodium acrylate 33% (w/v) | 8.6% (w/v) | |
| Acrylamide 50% (w/v) | 2.5% (w/v) | |
| N,N´-methylenebisacrylamide 2% (w/v) | 0.15% (w/v) | |
| 5 M NaCl | 11.7% (w/v) | |
| PBS | 1X |
Digestion buffer
| A | B | |
| Triton X-100 | 0.5% (w/v) | |
| EDTA 0.5 M, pH 8 | 0.2% (v/v) | |
| Tris-Cl 1 M, pH 8 | 5% (v/v) | |
| NaCl | 4.67% (w/v) | |
| proteinase K | 8 U/ml |
- 10% (v/v) normal goat serum
- 0.1% (v/v) Triton X-100
- PBS
- secondary antibody (Alexa Fluor, Invitrogen)
- Acryloyl-X SE solution (Thermo Scientific)
- 10% (w/v) TEMED
- 10% (w/v) APS stock solution
- poly-L-ornithine-coated coverslips
- acryloyl-X SE solution
- 0.5% (w/v) 4-hydroxy-TEMPO stock solutions
- Leica TCS SP8 confocal microscope (Leica, Germany)
- GraphPad Prism version 9.0.0 (RRID:SCR_002798)
Expansion microscopy
Expansion microscopy
6h
6h
Note
This protocol refers to the expansion microscopy (ExM) protocol described in Asano et al., 2018 with some modifications.
Block cells with 10% (v/v) normal goat serum (NGS) in 0.1% (v/v) Triton X-100 in PBS and incubate it with primary antibodies in blocking solution Overnight .
After a 3-h incubation with the corresponding secondary antibody (Alexa Fluor, Invitrogen), wash the samples and treat with 0.1 mg/ml Acryloyl-X SE solution (Thermo Scientific) in PBS for 03:00:00 at Room temperature .
3h
The freshly prepared gelling solution consisted of Stock X solution (8.6% (w/v) sodium acrylate 33% (w/v), 2.5% (w/v) acrylamide 50% (w/v), 0.15% (w/v) N,N´-methylenebisacrylamide 2% (w/v), 11.7% (w/v) NaCl 5 M, and PBS 1X), water, 10% (w/v) TEMED and 10% (w/v) APS stock solution in a 47:1:1:1 ratio.
Perform gel digestion Overnight in digestion buffer (0.5% (w/v) Triton X-100, 0.2% (v/v) EDTA 0.5 M, pH 8, 5% (v/v) Tris-Cl 1 M, pH 8, 4.67% (w/v) NaCl and 8 U/ml proteinase K).
3h
Add the gelling solution to each well and covered by a 15-mm coverslip to ensure the formation of a smooth, flat and thin gel.
Incubate coverslips for 01:00:00 at 37 °C for complete polymerization.
1h
Expand the gel in water for 01:00:00 and mount in 10 µg/mL poly-L-ornithine-coated coverslips to immobilize the gel for picture acquisition.
1h
Acquire images using a Leica TCS SP8 confocal microscope (Leica, Germany) equipped with a 100× /1.4 numerical aperture oil-immersion objective. For each condition, acquire 5 images from at least three independent experiments.
Analyze Images using Diffraction PSF 3D, DeconvolutionLab2, and EzColocalization plugins in Fiji-ImageJ.
Use GraphPad Prism version 9.0.0 (RRID:SCR_002798) to calculate Spearman’s rank correlation value (ρ) to identify colocalization of fluorescence signals.
The following is a variant of the protocol in case of using midbrain organoid sections:
Fix midbrain organoids and perform immunofluorescence staining as described above.
Treat sections with 0.1 mg/mL acryloyl-X SE solution in PBS at Room temperature Overnight .
Perform gelation in a 47:1:1:1 ratio of Stock X, 10% (w/v) TEMED, 10% (w/v) APS, and 0.5% (w/v) 4-hydroxy-TEMPO stock solutions.
Perform gel digestion and expansion as described above.
Acquire images using a Leica TCS SP8 confocal microscope (Leica, Germany) equipped with a 100× /1.4 numerical aperture oil-immersion objective. For each condition, acquire 5 images from at least three independent experiments.
Analyze Images using Diffraction PSF 3D, DeconvolutionLab2, and EzColocalization plugins in Fiji-ImageJ.
Use GraphPad Prism version 9.0.0 (RRID:SCR_002798) to calculate Spearman’s rank correlation value (ρ) to identify colocalization of fluorescence signals.